CSIRO PUBLISHING

www.publish.csiro.au/journals/app Australasian Plant Pathology, 2006, 35, 283–285

DISEASE NOTES OR NEW RECORDS

‘Candidatus Phytoplasma australiense’ is associated with diseasesof red clover and paddy melon in south-west Australia

M. SaqibA,C, M. G. K. JonesA,C and R. A. C. JonesA,B,C,D

AWA State Agricultural Biotechnology Centre, School of Biological Sciences and Biotechnology,Murdoch University, Perth, WA 6150, Australia.BPlant Pathology Section, Department of Agriculture, Locked Bag No. 4, Bentley Delivery Centre, WA 6983, Australia.CCentre for Legumes in Mediterranean Agriculture, University of Western Australia,35 Stirling Highway, Crawley, WA 6009, Australia.DCorresponding author. Email: rjones@agric.wa.gov.au

Abstract. Plants of red clover (Trifolium pratense), several other pasture legumes and paddy melon (Cucumismyriocarpus) with symptoms of diminished leaf size, pallor, rugosity, leaf deformation, shoot proliferation and stuntingwere observed amongst pasture plots growing at a site in south-west Australia. When tested using PCR, diseased samplesfrom all species with these symptoms were positive for a phytoplasma resembling ‘Candidatus Phytoplasma australiense’.This association was confirmed for red clover and paddy melon by subsequent nested PCR and sequence analysis. Thisis the first time that ‘Ca. Phytoplasma australiense’ has been reported infecting these hosts.

Additional keywords: Cucumis myriocarpus, hosts, perennial pasture legumes, Phytoplasma, Trifolium pratense.

Phytoplasmas are phloem-limited pathogens associated withmany damaging plant diseases (Seemuller et al. 1998).One of the phytoplasma species most commonly foundin Australia is ‘Candidatus Phytoplasma australiense’. InAustralasia, it is associated with diseases of several hostsincluding cabbage tree (Cordyline australis), common bean(Phaseolus vulgaris), papaya (Carica papaya), pumpkin(Cucurbita maxima), strawberry (Fragaria × ananassa),grapevine (Vitis vitifera), and chickpea (Cicer arietinum)(Liefting et al. 1998; Padovan et al. 1999, 2000; Andersenet al. 2001; Streten et al. 2005; Saqib et al. 2005). Insouth-west Australia, it causes a damaging disease ofPaulownia (Paulownia fortunei) trees (Bayliss et al. 2005).There are records of phytoplasma-like diseases in awide range of other cultivated plants in this regionbut without molecular identification of the causal agent.The host species involved include aster (Callistephuschinensis), bird of paradise plant (Poinciana gilliesii), carrot(Daucus carota), cape gooseberry (Physalis peruviana),celery (Apium graveolens), dahlia (Dahlia spp.), geranium(Pelargonium spp.), gerbera (Gerbera jamesoni), larkspur(Delphinium ajacis), lettuce (Lactuca sativa), lucerne

(Medicago sativa), michaelmas daisy (Aster novae-angliae),marigold (Calendula officinalis), periwinkle (Vinca spp.),petunia (Petunia hybrida), phlox (Phlox drummondii),potato (Solanum tuberosum), shasta daisy (Chrysanthemummaximum) and snapdragon (Antirrhinum majus) (Doepel1964; Goss 1964; McLean and Price 1984).

In September 2004, symptoms of diminished leaf size,pallor, rugosity, leaf deformation, shoot proliferation andsevere stunting were observed in plants of red clover(Trifolium pratense) within plots containing otherwisevigorously growing plants (Fig. 1a). These plots were at theMedina Research Station just south of Perth, and belongedto a program selecting perennial pasture species suitedto south-west Australian conditions. Symptomatic plantswere dug out, potted, kept in insect-proofed glasshouses atabout 20◦C, and used as tissue sources for serological andPCR tests.

The possibility that the disease was of viral origin wasinvestigated first. Symptomatic red clover leaf samples weretested by enzyme-linked immunosorbent assay (ELISA)or tissue blot immunosorbent assay (TBIA) as describedby Coutts and Jones (2000) using antibodies to the

© Australasian Plant Pathology Society 2006 10.1071/AP06012 0815-3191/06/020283

284 Australasian Plant Pathology M. Saqib et al.

(a)

(b)

Fig. 1. Phytoplasma-affected plants in the field. (a) Symptomatic redclover plant, left foreground, with three much larger asymptomatic redclover plants. (b) Paddy melon plant showing diminished leaf size, leafchlorosis, proliferation of shoots, and stunted growth habit.

following viruses previously found infecting pasture legumesin the region: Alfalfa mosaic virus (genus Alfamovirus,family Bromoviridae), Bean yellow mosaic virus (genusPotyvirus, family Potyviridae), Cucumber mosaic virus(genus Cucumovirus, family Bromoviridae), Beet westernyellows virus (genus Polerovirus, family Luteoviridae) andSubterranean clover mottle virus (genus Sobemovirus, familyunassigned); with antibodies to Tomato spotted wilt virus(genus Tospovirus, family Bunyaviridae); and with a genericantibody to members of the Luteoviridae. The results wereall negative. Samples were then sent to Dr Safaa Kumari atICARDA, Syria who used TBIA to test them with antibodiesto the following additional viruses: Broad bean wilt virus(genus Fabavirus, family Comoviridae), Chickpea chloroticdwarf virus (genus Mastrevirus, family Geminiviridae), Fababean necrotic yellows virus and Subterranean clover stuntvirus (both genus Nanovirus, family Nanoviridae). Thesetests were again negative. Also, sap inoculation tests usingleaf extracts from symptomatic red clover plants failedto cause any infection in a range of standard plant virusindicator hosts.

In May 2005, the site was revisited and symptomaticplants were again found in red clover plots. Similar symptomswere also observed in plots of other species of pasturelegumes, including alsike clover (Trifolium hybridum),Glycine canescens, Lablab purpureus and Lotononis bainseii.In addition, some plants of the common cucurbitaceous weedpaddy melon (Cucumis myriocarpus) (McKenzie et al. 1988)were found with symptoms of reduced leaf size, pallor, leafdeformation and proliferation of shoots (Fig. 1b). Plants of allthese species with symptoms were dug up, potted, maintainedin a glasshouse and kept as a source of tissue samples forfurther tests.

The possibility that the symptoms observed in perennialpasture legume species and paddy melon at the Medina sitemight be caused by a phytoplasma rather than a virus was theninvestigated. Total DNA was extracted from symptomatic andasymptomatic control plants of red clover, paddy melon andthe other perennial pasture legumes by the hot CTAB methodwith minor modifications (Dellaporta et al. 1983; Zhang et al.1998. Primers P1 (Deng and Hiruki 1991) and P7 (Schneideret al. 1995a) were used to amplify the 1.8-kb PCR productof the 16S rRNA gene from the genomic DNA, followed bynested primers R16mF2 and R16mR1 (Gundersen and Lee1996; Schneider et al. 1995b) to amplify about 1200-bp PCRproducts from the initial 16S rRNA amplified PCR product.Amplified fragments were analysed by gel electrophoresis(Fig. 2). All symptomatic samples gave amplified bands.The amplified PCR products from red clover and paddymelon were purified using a Clean-up Kit (GeneWorks),ethanol-precipitated and the pellets re-suspended in TEbuffer. The 1200-bp products amplified by the nested primerswere sequenced directly using primer R16mF2, Big DyeTerminators (Perkin-Elmer, Foster City, CA, USA), and anApplied Biosystems 3730 sequencer. PCR and sequencing

M 1 2 3 4 5 6 7

1200 bp

Fig. 2. 1% agarose gel showing PCR products amplified from sampletissues. M: 1 kb DNA marker; lane 1: healthy red clover sample;lanes 2–6: red clover samples collected from different diseased plants;lane 7: sample from diseased paddy melon.

Ca. Phytoplasma australiense in red clover and paddy melon Australasian Plant Pathology 285

was done twice. These sequences were analysed using thecomputer program Seq Ed 1.0.3, and then compared withthose of known phytoplasma origin in the GenBank database.These sequences from red clover and paddy melon (GenBankreferences DQ269471 and DQ310885, respectively) shared100% homology with each other and 99% homology withtwo phytoplasmas associated with either dieback or yellowcrinkle and mosaic diseases of papaya (GenBank referencesY10096 and Y10097; White et al. 1998), and also withalfalfa witches-broom phytoplasma reported from Oman(GenBank reference AY169323). These sequencing resultsconfirmed the presence of the ‘Candidatus Phytoplasmaaustraliense’ in the diseased red clover and paddymelon samples.

The plots with symptomatic plants had been growing for 2or more years and many plants were still asymptomatic. Thedisease was, therefore, spreading slowly. The symptoms weresevere in affected plants, and so symptomatic plants shouldbe rogued out to diminish infection sources for spread tonearby plants or plots. Further research is needed to identifythe insect vector and establish which additional alternativehosts are important as reservoirs of infection other than paddymelon. Further research is also needed to establish the causalagents of the many other phytoplasma-like diseases that occurin south-west Australia (McLean and Price 1984).

Acknowledgements

We thank Dr Safaa Kumari for serological tests on samples,Tracey Smith and Eva Gajda for technical support, and theWestern Australian Perennial Pasture Improvement Programfor permitting us to sample their plots and providinginformation on the species being grown.

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Accepted 10 November 2005

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