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www.publish.csiro.au/journals/app Australasian Plant Pathology, 2006, 35, 293–296

Identification of sweet potato little leaf phytoplasma associatedwith Vigna unguiculata var. sesquipedalis and Lycopersicon esculentum

M. SaqibA,B, K. L. BaylissB and M. G. K. JonesA,B,C

AWA State Agricultural Biotechnology Centre, Murdoch University, Perth, WA 6150, Australia.BSchool of Biological Sciences and Biotechnology, Murdoch University, Perth, WA 6150, Australia.

CCorresponding author. Email: m.jones@murdoch.edu.au

Abstract. Vigna unguiculata var. sesquipedalis (snakebean) and Lycopersicon esculentum (tomato) plants withphytoplasma-like symptoms were found in the horticultural region in Broome, Western Australia. The symptomson snakebean included ‘witches’ broom’ and proliferation of small leaves, and on tomato they varied from phyllodyand ‘big bud’ to ‘witches’ broom’ and proliferation of small leaves. Using universal primers for phytoplasmas, thecomplete 16S rRNA gene and intergenic spacer region, and part of the 23S rRNA gene were amplified by PCR.Sequence analysis identified that the agent associated with the symptoms was a strain of sweet potato little leaf strainV4 (SPLL-V4) phytoplasma (16SrII group, strain of ‘Candidatus Phytoplasma australiense’). SPLL phytoplasmahas not been reported before in snakebean or tomato in this isolated agricultural region.

Additional keywords: Broome, ‘little leaf’, phyllody, tomato big bud, witches’ broom.

IntroductionIn a survey of plant pathogens in crop plants in north-western Australia, Vigna unguiculata var. sesquipedalis(snakebean) and Lycopersicon esculentum (tomato) plantswith phytoplasma-like symptoms were found at propertiesnear Broome in Western Australia. Phytoplasmas are phloem-limited plant pathogens that are associated with many plantdiseases, and which contribute to economic losses in crops(McCoy et al. 1989). The most widespread phytoplasma ofcrop plants in northern Australia is tomato big bud (TBB)phytoplasma (Davis et al. 1997). In isolated agriculturalareas, such as in the vicinity of Broome, phytoplasmainfections in crops probably originate in native vegetation(Hutton and Grylls 1956), but could also be introduced indiseased stock.

The method widely used for the classification anddifferentiation of phytoplasmas is analysis of 16S rRNAgene sequences (Seemuller et al. 1994). Restriction analysisand DNA sequencing of the 16S rRNA gene can beused to differentiate between TBB phytoplasma and otherphytoplasmas such as sweet potato little leaf strain V4 (SPLL-V4) phytoplasma (Padovan et al. 2000a). SPLL phytoplasmawas first detected in Australia in Ipomoea batatas(Convolvulaceae) in the Northern Territory (Gibb et al.1995). Phytoplasma infections, such as TBB phytoplasmain Emilia sonchifolia and SPLL phytoplasma in Cucurbita

maxima (Japanese pumpkin), have been reported in theBroome agricultural area (Schneider et al. 1999). This paperextends the host range of SPLL phytoplasma in Australia toinclude snakebean and tomato.

MethodsPlant material

Leaf and stem samples of plants of snakebean and tomato showingsymptoms including ‘witches’ broom’, ‘little leaf’, ‘big bud’ andphyllody were collected from different locations in Broome andKununurra, in the north of Western Australia. The samples were storedat 4◦C before grafting or further processing. Symptomatic parts ofsnakebean and tomato were side-and tip-grafted onto V. unguiculata var.unguiculata (cowpea) (Fig. 1b, c) and tomato (cv. Grosse Lisse)(Fig. 1 f, g ) plants, respectively, and maintained in an air-conditionedglasshouse at 20–24◦C.

DNA extraction

The hot CTAB method (Dellaporta et al. 1983; Zhang et al. 1998) wasused with minor modifications to extract the DNA from snakebean andtomato tissues. One gram of green leaf tissue was frozen in liquidnitrogen and ground to a fine powder with a mortar and pestle. Thematerial was transferred to a 50-mL centrifuge tube and 15 mL ofcetyltrimethylammonium bromide (CTAB) buffer was added at 60◦C(CTAB buffer: 2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris–HCl,pH 8.0, 0.2% β-mercaptoethanol). The mixture was incubated in a 60◦Cwater bath for 20 min and then placed on ice for 5 min. An equal volumeof phenol:chloroform:isoamyl alcohol mixture (24 : 24 : 1) was addedand the sample was mixed vigorously and centrifuged for 15 min at

© Australasian Plant Pathology Society 2006 10.1071/AP06028 0815-3191/06/030293

294 Australasian Plant Pathology M. Saqib et al.

(a) (b)

(c) (e)

(d )

(f ) (g)

Fig. 1. Field and glasshouse symptoms of infected plants. (a) Infected snakebean in the field. (b) Symptomaticsnakebean stem tissue grafted on cowpea in the glasshouse. (c) Transmission of symptoms from snakebean bytip-grafting to cowpea (left) with healthy control cowpea plant (right). (d ) Tomato plant in the field showingwitches’ broom and little leaf symptoms. (e) Infected tomato in field showing symptoms of big bud and phyllody.( f ) Tomato plant (cv. Grosse Lisse) in the glasshouse showing witches’ broom and little leaf symptoms aftergrafting with affected tomato plants from the field. (g) Tomato plant (cv. Grosse Lisse) in glasshouse graftedwith infected tissues showing big bud and phyllody-like symptoms (left) and healthy tomato (right).

Identification of sweet potato little leaf phytoplasma Australasian Plant Pathology 295

30 070 g. The supernatant was transferred to a new 50-mL centrifugetube and mixed with an equal volume of ice-cold isopropanol and leftovernight at −20◦C to precipitate DNA. The tube was then centrifugedat 30 070 g for 15 min, the pellet rinsed in 70% ethanol, dried, andresuspended in 1 mL of Tris-EDTA (10 mM Tris–HCl, 1 mM EDTA,pH 8.0), and the DNA was stored at −20◦C.

PCR conditions and product treatment

PCR was performed using primers P1 (Deng and Hiruki 1991)and P7 (Schneider et al. 1995) to amplify a 1.8-kb product of the16S rRNA gene, the complete 16S–23S rRNA intergenic spacerregion and part of 23S rRNA gene of phytoplasmas from the DNAof symptomatic tomato, snakebean and snakebean-grafted cowpeaplants (Schneider et al. 1999). PCR cycling conditions were 94◦Cfor 1 min followed by 35 cycles of 94◦C for 1 min, 56◦C for 1 min,and 72◦C for 1.5 min. To purify PCR products, a PCR Clean-up Kit(GeneWorks, Adelaide, South Australia) was used. Purified PCRproducts were ethanol-precipitated and the pellet was resuspended inTE buffer. Reference 16S rRNA DNA of Paulownia witches’ broomphytoplasma (PaWB), Australian grapevine yellows phytoplasma(AGY) and TBB phytoplasma were kindly supplied by Ing-MingLee (USDA), Fiona Constable (DPI Victoria) and Karen Gibb(Charles Darwin University), respectively, and were included aspositive controls.

Cloning and sequence analysis

Amplified products from symptomatic tomato, snakebean and cowpeaplants were sequenced directly using primers P1 and P7. PCR productsfrom snakebean were cloned using the pGEM-T easy vector andJM109 competent cells (Promega, Madison, WI, USA). The clonedinserts were sequenced using T4 and SP6 primers. Sequencingwas performed with Big Dye Terminator 3.1 (Perkin-Elmer, FosterCity, CA), with an Applied Biosystems 3730 sequencer. PCR andsequencing was repeated three times to check for the possibility ofmixed infections. The 1.8-kb nucleotide sequences were analysedusing the computer program Seq Ed 1.0. 3 and the sequences fromtomato (DQ375778) and from snakebean (DQ375777) were submittedto GenBank and compared with those of known phytoplasmas in theGenBank database.

Results

The symptomatic plants collected in Broome were snakebeanand tomato, and the occurrence of the symptoms waswidespread. The symptoms of infection of the phytoplasmasin the field and after grafting onto cowpea and tomato inthe glasshouse are shown in Fig. 1; field and glasshousesymptoms were similar. In both cowpea and tomato, therewere different morphologies. For cowpea, these were multiplestem branching (witches’ broom) and a proliferation of smallleaves (little leaf), and for tomato, the symptoms were largerbuds (big bud), with hardened stems, phyllody, purple stemand bud colouration, and witches’ broom.

PCR products of the expected size (1800 bp) wereamplified using P1 and P7 primers from all field and graftedsamples, and from control TBB phytoplasma DNA (Fig. 2).The 16S rRNA gene sequences of the amplified region(excluding the spacer region) were compared with eachother and with other sequences in GenBank. Those frominfected snakebean and tomato all showed 100% similarity

M 1 2 3 4 5 6 7

2 kb

Fig. 2. One percent agarose gel of PCR products amplified fromsymptomatic plants showing diagnostic bands of 1.8 kb. M: 1 kb DNAsize marker (Fermentas); lane 1: affected leaf of snakebean; lane 2:tissue from cowpea grafted with symptomatic snakebean; lanes 3–5:tissue from tomato grafted with symptomatic field tomato; lanes 6–7:negative controls of healthy cowpea and tomato, respectively.

with each other, 99% similarity with the 16S rRNA geneof SPLL strain V4 (SPLL-V4) phytoplasma (AJ289193)isolated from Catharanthus roseus and 98% similaritywith that of TBB phytoplasma (Y08173). These dataindicate that the agent associated with symptoms includingwitches’ broom, little leaf, big bud and phyllody fromboth cowpea and tomato is most closely related to theSPLL-V4 phytoplasma.

Discussion

The work described here was part of a survey of plantviruses and phytoplasmas undertaken in the agriculturalregions at Broome and Kununurra in the north-west ofWestern Australia. In previous surveys in northern Australia,TBB phytoplasma was found to be relatively widespread,and has been reported to infect both tomato and legumesincluding cowpea, V. luteola and V. trilobata (Davis et al.1997). An unknown phytoplasma has also been reported inV. unguiculata var. diakinditata in Queensland (Davis et al.1997) and vigna little leaf phytoplasma is associated withlittle leaf symptoms in V. lanceolata in the Northern Territory(Schneider et al. 1999). In our work, we initially expectedthat the symptoms found would also be associated withTBB phytoplasma, but our sequencing results indicate thatthe phytoplasma associated with the symptomatic plantsis more likely to be a strain of SPLL-V4 phytoplasma.Some minor differences between sequences might also be

296 Australasian Plant Pathology M. Saqib et al.

associated with incorporation errors occurring during PCR.Although there was some variation in disease symptomsbetween different infected plants and some differences insymptoms were noted between side- and tip-grafted plants,the PCR and sequencing results provided no evidencefor mixed infections of phytoplasmas. The differences insymptoms are probably related to slightly different responsesto infection by different genotypes of host plant (Padovanet al. 2000b). There could also be sequence differencesin genes other than the 16S rRNA gene that might reflectvariation in the pathogen related to differing symptoms(Streten and Gibb 2003).

There have been several recent reports on phytoplasma-associated diseases of crops newly introduced into Australia,such as the incidence of phytoplasma in Paulowniaplantations in the south-west of Western Australia(Bayliss et al. 2005), or in new hosts such as chickpeain Kununurra (Saqib et al. 2005), red clover (Trifoliumpratense) and paddy melon (Cucumis myriocarpus) insouth-western Australia (Saqib et al. 2006), and capsicumand celery in Queensland (Tran-Nguyen et al. 2003).SPLL-V4 phytoplasma infections of snakebean and tomatohave not been reported before in the Broome area, orin snakebean as a host. The increased recognition ofphytoplasma infection of crop plants, particularly innorth-western Australia, suggests that there is a need forfurther study of the economic and ecological effects ofphytoplasma-associated diseases.

Acknowledgements

This work was supported by a Murdoch University ResearchExcellence Grants Scheme (REGS) grant to M. G. K. Jones.We thank the reviewers for useful comments and suggestions.

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Received 15 August 2005, accepted 13 November 2005

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