First report of Phoma clematidina the cause of leaf spot-wilt disease of Clematis pubescens in Australia
Leaf lesions and wilt of Clematis pubescens were observed in the Jarrah (Eucalyptus marginata) forest of South-Western Australia in 2009. A Phoma sp. was consistently isolated from the infected tissues and its morphological characteristics were determined. The identity of the fungus was confirmed by sequencing of the internal transcribed spacer (ITS) region of the rDNA operon and Phoma clematidina was identified. A pathogenicity test was conducted and Koch’s postulates were fulfilled by re-isolation of the identified fungus. This is the first report of P. clematidina causing leaf lesions and wilt of Clematis pubescens in Australia.
KeywordsPhoma leaf spot-wilt Clematis Rehabilitation Jarrah forest
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).
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.
The authors would like to thank Ms Paula Mather for technical assistance.