The cosmopolitan genus Clematis is a member of the Ranunculaceae family and contains 300 species, mostly climbers or groundcovers. Many of these are grown commercially. Clematis pubescens Endl. is found throughout the Jarrah (Eucalyptus marginata) forest of South-Western Australia. There are three Clematis species present in Western Australia (C. delicata, C. linearifolia, C. pubescens) but only C. pubescens occurs in the northern Jarrah forest (Wheeler et al. 2002).

Alcoa of Australia Limited operates two bauxite mines in the northern Jarrah forest and about 600 ha are mined and rehabilitated annually. The rehabilitation aim is to restore a self-sustaining Jarrah forest ecosystem (Elliot et al. 1996; Gardner 2001). Due to low germination and survival rates, it is difficult to establish C. pubescens using broadcast seed in rehabilitation areas. The current practice involves planting established seedlings and has been especially developed for rehabilitation of bauxite mines by Alcoa’s Marrinup Nursery.

As C. pubescens is new to horticultural production, little is known about its diseases although sporadic leaf lesions were observed in 2008. Ascochyta clematidina and Coniothyrium clematidis-rectae have been reported as the causal organisms of leaf spot and wilt of Clematis spp. However, A. clematidina was transferred to the genus Phoma as Phoma clematidina (Thüm.) Boerema, Versl. Meded. Plziektenk. (Woudenberg et al. 2009). P. clematidina causes destructive disease on Clematis spp. and is widespread in Europe, America and New Zealand (Smith and Cole 1991; van de Graaf et al. 2001).

Symptoms of disease caused by P. clematidina were observed on C. pubescens in the south-west of Western Australia in 2009 (Fig. 1). Typical symptoms appeared on the leaves of propagated plants at the nursery as purple lesions that eventually coalesced to form large necrotic areas (Fig. 2). Favourable growing conditions during autumn 2009 resulted in the propagated plants having excessively long above-ground stems; too long for carrying into the mine rehabilitation sites during the forthcoming rainy season (June to August). To facilitate the transport and transplanting of the propagated plants, the above-ground stems were heavily pruned near the end of the dry season, which was a new cultural practice and has been rarely adopted previously. The outbreak of leaf spots and wilt were pronounced on C. pubescens after transplanting. Pruning appeared to have increased the risk of infection via wounded tissues. P. clematidina has been reported to be a wound pathogen causing leaf spots on both wilt susceptible and wilt resistant cultivars (Smith and Cole 1991).

Fig. 1
figure 1

Clematis pubescens showing leaf lesions and wilt after transplanting in the mine re-vegetation site of the south–west of Western Australia

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Fig. 2
figure 2

Leaf lesions on Clematis pubescens (left) caused by Phoma clematidina on the propagated plants in the nursery (right)

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Twenty five sections of plant tissue surrounding the leaf lesions were surface-sterilised by immersion in a 1.25% aqueous solution of sodium hypochlorite for 2 min, rinsed in sterile water and dried in a laminar flow cabinet. The sterilised leaf pieces were then either placed on potato dextrose agar (PDA) and incubated at 22 ± 3°C, with developing fungal colonies sub-cultured onto PDA and then single-spored to obtain pure cultures; or placed in trays on moist filter paper and incubated at 25°C with a 12-h dark and light cycle. A Phoma sp. was consistently isolated and the colony size, colony morphology and morphological characteristics of the isolated fungus were determined for 10 isolates. The colony was white to pale grey turning olivaceous grey with age. The average colony size was 30–40 mm after 7 days on PDA. Chlamydospores were uni- or multicellular thick-walled, brown, guttulate, in chains and 6–10 μm (unicellular) or 25–40 × 10–25 μm (multicellular) in diam. Pycnidia were immersed in culture and were formed subepidermally in leaf lesions. They were dark brown, subglobose, ostiolate and 100–125 μm in diam. Conidia were unicellular, variable in shape, subellipsoidal to cylindrical, usually 5–9 × 2–3.5 μm, occasionally larger with 1 septa and 10–15 × 3–4.5 μm on malt-extract agar (MEA) (Fig. 3a). However, conidia were distinctly large (12–25 × 4.5–6.5 μm) with up to two septa in older pycnidia that formed on the leaf lesions (Fig. 3b). Both cultural and morphological characteristics of the fungal isolates were similar to those described for P. clematidina (Boerema et al. 2004; Woudenberg et al. 2009).

Fig. 3
figure 3

Conidia of Phoma clematidina in vitro (a) and in vivo (b). Bars = 5 μm

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Two representative isolates of morphologically identified P. clematidina were grown on PDA for 2 weeks at 25°C. Total genomic DNA was extracted from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen, Melbourne, Vic., Australia) according to the manufacturer’s instructions. Amplification of the internal transcribed spacer (ITS)1 and ITS2 regions flanking the 5.8S rRNA gene were carried out with universal primers ITS1 and ITS4 according to the published protocol (White et al. 1990). The polymerase chain reaction (PCR) product was purified with Qiagen PCR purification kit (Qiagen, Melbourne, Vic Australia) and sequenced on an ABI 3730 DNA Sequencer (Applied Biosystems, Melbourne, Vic., Australia) at Murdoch University, Perth, WA, Australia. The two sequences of the ITS1 and ITS2 regions were identical to the sequences from the same region of P. clematidina (GenBank Accession No. FJ426991) in the NCBI database (www.ncbi.nlm.nih.gov/) as published by Aveskamp et al. (2009).

Pathogenicity tests were performed on 2-month old C. pubescens plants using two representative isolates of P. clematidina. Leaves and stems of three plants were inoculated by either wounding or non-wounding per isolate. Wounding was used as P. clematidina has been reported to be a wound pathogen (Smith and Cole 1991). A conidial suspension (106 spores/mL) was prepared in sterile water using a culture of each isolate grown on MEA. For the wounding inoculation test, the conidial suspension was dropped on the leaf and stem sites (5 μL per site/10 sites per plant) after pin-prick wounding using a syringe needle. For the non-wounding inoculation test the conidial suspension was sprayed on the plant surface to runoff. Control plants (one wounded and one non-wounded) were inoculated using sterilised water. All plants were placed under mist for 48 h and then moved to a growth room chamber at 22 ± 3°C. Leaf lesion and wilt symptoms were observed 2–3 weeks post-inoculation. The symptom development was faster and more pronounced on wound-inoculated plants. Control plants remained asymptomatic.

Koch’s postulates were fulfilled by re-isolation of P. clematidina. A culture of P. clematidina, the morphological identity of which was confirmed via ITS sequencing, was deposited in the Western Australia Plant Pathogen Collection (WAC13312). To our knowledge, this is the first report of P. clematidina causing leaf spot-wilt disease on C. pubescens in Australia.