Australasian Plant Pathology (1999) 28: 108-]]4

Genetic variability in phytoplasmas associated with papaya yellow crinkle and papaya mosaic diseases in Queensland and the Northern Territory S.J. De La Rue, B. Schneider and KS. Gibb School of Biological and Environmental Sciences, Northern Territory University, Darwin, Northern Territory 0909 Australia Corresponding author: SJ. De La Rue (Email [email protected])

Abstract Samples of papaya (Carica papaya L.) showing symptoms of papaya yellow crinkle (PYC) or papaya mosaic (PM) diseases were collected from two study sites in Queensland (Rockhampton and Caboolture) and from Katherine in the Northern Territory. Phytoplasmas were detected in these samples using polymerase chain reaction (PCR) and fluorescence microscopy techniques. The genetic variability of the phytoplasmas was studied using Southern blot hybridisation, restriction fragment length polymorphism (RFLP), and sequence analysis. RFLP analysis of PYC samples collected from Caboolture indicated that only the tomato big bud (TBB) type phytoplasma was present, whereas both PYC and PM samples collected from Rockhampton exhibited two phytoplasma types, sweet potato little leaf variant (SPLL- V4) and TBB. PYC samples collected from Katherine showed even greater variability with cactus witches' broom (CWB), which was first described from Indonesia, detected in addition to SPLL- V4 and TBB which are common throughout Australia. Although PYC and PM diseases have different symptoms, no genetically distinct phytoplasma could be found in exclusive association with either disease.

Additional keywords: PCR, RFLP

Introduction Papaya mosaic (PM) disease has only recently been associated with a phytoplasma (Gibb et al. 1996b; Liu et at. 1996; White et at. 1997), whereas phytoplasmas have been implicated in papaya yellow crinkle (PYC) disease for some time (Gowanlock et at. 1976). PYC causes the older leaves to turn yellow and droop after which the crown leaves develop translucent areas around the edges and main veins which eventually become necrotic, making the leaves look tattered and claw like. Fruit production is affected by the development of virescence (greening of floral parts) and phyllody (the changing of floral parts into leaves). Plants affected with PM have yellow and stunted young leaves which have translucent areas around the margin and often develop many mosaic-affected side shoots. The main distinguishing feature of PM is the presence of water-soaked lesions on the stem, petioles and fruit 108

ofaffected plants whereas PYC can be distinguished by floral abnormalities such as phyllody and virescence (Peterson et al. 1993). Although the symptoms ofPYC and PM do differ, restriction fragment length polymorphism (RFLP) analysis ofa ribosomal DNA fragment amplified by polymerase chain reaction (PCR) indicated that the PYC and PM phytoplasmas were genetically similar to each other and to TBB (Gibb et al. 1996b). These phytoplasmas are in the faba bean phyllody cluster which is the most prevalent group in Australia (Davis et at. 1997). This study examines in more detail the genetic diversity ofthe phytoplasmas associated with PYC and PM from study sites in the Northern Territory and Queensland. As in the study by Gibb et al. (1996b), all the samples were initially compared with RFLP analysis of a ribosomal fragment amplified by PCR using universal primers. Samples showing different RFLP patterns were then compared by Australasian Plant Pathology Vol. 28 (2) 1999

sequence analysis of the spacer region between the 16S and 23S rRNA genes. In addition to comparisons ofthe ribosomal genes, Southern blot hybridisations were performed using randomly cloned sweet potato little leaf variant V4 (SPLLV4) DNA as probes.

Methods Source of material Seven PYC samples were obtained from Caboolture (CI to C7), Queensland (D. Persley, Department of Primary Industries (DP!), Brisbane). Eleven samples were obtained from Rockhampton (R 1 to R 11), Queensland (R. Elder, DPI, Rockhampton), of which six were identified in the field as PYC and five as PM. Samples were diagnosed as PM on the basis of watersoaked lesions on the fruit and petioles. A further eight PYC samples were collected from Katherine (K1 to K8), Northern Territory (R. Davis, Northern Territory University, Darwin), of which five were provided as PCR product only. Reference strains The sweet potato little leaf (SPLL) phytoplasma cultured in sweet potato, the SPLL variant V4 (SPLL- V4) phytoplasma in periwinkle (Catharanthus roseus (L.) G. Don) and the tomato big bud (TBB) phytoplasma also cultured in periwinkle were used as reference isolates (Davis et al. 1997). These three phytoplasmas were maintained in their respective hosts by periodic grafting onto young plants. DNA extraction The midribs and petioles ofpapaya samples from Queensland were cut into 10 mm portions and Iyophilised. Total DNA was extracted for PCR and Southern blot hybridisations using the method described by Doyle and Doyle (1990). For the Katherine samples, DNA was extracted from fresh midrib and petiole tissue using the method of Ahrens and Seernuller (1992). DNA quality and quantity were checked by agarose electrophoresis. This DNA was subsequently used in PCR experiments. Phytoplasma detection with fluorescence microscopy Papaya midribs were sectioned with a freezing microtome and stained with the DNA fluorochrome DAPI (Seernuller 1976). Phytoplasmas were visualised with a fluorescence microscope. Australasian Plant Pathology Vol. 28 (2) 1999

Polymerase chain reaction (peR) PCR was done using primers PI (Deng and Hiruki 1991) and m23sr (Padovan et at. 1995) to give a product of approximately 1700bp. These primers amplifythe 16SrRNA gene, the spacer region, and the beginning of the 23S rRNA gene. PCRreactions contained 50-1 00 ng of DNA, 0.2 mM of each dNTP, 0.4 J..lM of each primer, 1 unit ofthermostable Taq DNA polymerase (Advanced Biotechnologies Ltd., Surrey, UK), and I x DNA polymerase buffer (supplied with the enzyme) in a [mal volume of 50 J..lL. After manual hot start, the samples were subjected to 35 amplification cycles consisting of95°C for 60 sec, 55°C for 60 sec, and noc for 90 sec. Amplifications were done in a Corbett FTS-320 thermocycler (Corbett Research, Mortlake, New South Wales, Australia). The presence of a PCR product was determined after gel electrophoresis of 5 J..lL of reaction mix on a 1% agarose gel in 0.5 x TBE buffer (45 J..lM Tris-borate, 1 mM EDTA, pH 8.0). If inhibition was suspected, the PCR was repeated using a 1: 10 dilution of template DNA in water. Southern blot analysis DNA (10-20 ug) extracted from Iyophilised plant material was digested with either EcoRI (80 units), or HindIII (80 units), or double digested with 40 units of each restriction enzyme (New England Biolabs, Beverly,MA, USA). The samples were digested overnight at 37°C, and the DNA then ethanol precipitated (Sambrook et at 1989) and resuspended in a minimal volume to allow all the digested DNA to be loaded into the wells of a 1% agarose gel for fragment separation by electrophoresis in 0.5 x TBE. The DNA was transferred to a nylon membrane (Hybond N+, Amersham, Baulkham Hills, New South Wales, Australia) as described by Sambrook et at (1989). The DNA was bound to the membrane by UV cross linking. The random SPLL-V4 clones pH4 (1.7 kb), pH30 (1.0 kb) and pH80 (2.7 kb) provided by B. Schneider (Northern Territory University, Darwin) were used in a cocktail as described by Davis et al. (1997) and labelled with a 32P-dATP using the Prime-a-gene labelling system (Promega Corporation, Sydney, New South Wales, Australia) according to the manufacturer's instructions. Samples digested with HindIII and EcoRIJHindIII were hybridised with the cocktailprobe, and samples digested with EcoRi were hybridised with the SPLL- V4 probe pE21 (1.6 kb) also provided by B. Schneider. Membranes were prehybridised for at least 3 h at 55°C in 30 mL of prehybridisation solution (Sambrook et at 1989). 109

After prehybridisation, the solution was removed and replaced with 3 mL of hybridisation solution containing the denatured radio labelled probe. Hybridisation was then allowed to continue overnight at 55°C. After hybridisation, the membranes were washed twice for 5 min at room temperature with 2 x SSC and 0.1% SDS, followed by two 30 min washes at 55°C with 0.2 x SSC and 0.1% SDS. The membranes were exposed to X-ray film with intensifying screens at -75°C for 24--48 h. Restriction fragment length polymorphism (RFLP) analysis PI/m23sr PCR products (5 flL) were digested with either AZul, Rsai or estEll (New England Biolabs, Beverly, MA, USA) according to the manufacturer's instructions. To ensure complete digestion, reaction mixtures were incubated either overnight at 37°C with either AZul or RsaI, or at 60°C for 2 h with BstEII. The AZul and RsaI fragments were separated by electrophoresis in I x TBE using 8% and 5% polyacrylamide gels, respectively. The estEll fragments were separated using 1.5% agarose gels in 0.5 x TBE. Gels were stained with ethidium bromide (0.3 ug/ml.) and observed under UV illumination. Sequencing PCR products of the samples for sequencing were purified with the Wizard DNA Clean-up system (Promega Corporation, Sydney, New South Wales, Australia). The intergenic spacer region of the PCR products was then manually sequenced using primers P3 (Schneider et at. 1995) and m23sr and the AmplrCycle sequencing kit (Perkin Elmer, Norwalk, CT, USA) with a_33p dATP used according to manufacturer's recommendations for the incorporation method. Electrophoresis ofthe sequencing reactions was done using a 5% acrylamide gel. Thermocycling parameters were as follows: 30 cycles of I min at 95°C, I min at 45°C, and I min at 72°C. Sequence analyses were done using programs housed at the Australian National Genomic Information Service (University of Sydney, New South Wales, Australia). A dendrogram derived from alignments based on pairwise similarity scores (gap weight 5.0, gap length weight 0.5) was done using PileUp (GCG University of Wisconsin) . Sequences used in the analysis were obtained from the EMBL and GenBank databases.

110

Results Phytoplasma detection using PCR and fluorescence microscopy Ofthe 26 papaya samples used for this study (Table 1),24 were phytoplasma positive by PCR. Phytoplasma DNA was observed by fluorescence microscopy in 14 of the 21 samples tested. Only one of these 14 samples was not amplified by PCR (Rll-YC). DNA was not amplified from healthy papaya DNA or water (results not shown). Determination of genetic relatedness between PYC and PM using Southern blot hybridisation SelectedsamplesfromRockhampton (Rl-YC, R2-M, R3-M, R4-M andR5-YC) and Caboolture (C2-YC, C5-YC and C6-YC) digested with EcoRI and/or HindIII were used for Southern blot hybridisation experiments using the cocktail probe ofpH4, pH30 and pH80. All samples gave the same fragment pattern with no discernible differences between PYC and PM (results not shown). Similarly the papaya samples digested with EcoRI and probed with the SPLL- V4 clone pE21 were identical (results not shown). In all Southern blot hybridisations the fragment patterns from the papaya phytoplasmas were the same as that of the SPLL-V4 reference phytoplasma and at no time was a positive signal observed from healthy papaya DNA. Detection of genetic variants with RFLP RFLP analysis of the ribosomal gene amplified by PCR with AZuland Rsai showed that all the samples from Caboolture (Figures lA and 2A) and the majority of the samples from Rockhampton (Figures lB and 2B) were of the TBB type, except for sample R2- M (Figures IB and 2B, lane 3) which was a SPLL-V4 RFLP type. The samples from Katherine (Figures 1C and 2C) showed a high degree ofvariability with three fragment patterns observed, the TBB and the SPLL-V4 patterns and one unique pattern (Figure IC and 2C, lanes 8 and 9, K7-YC and K8-YC, respectively). These results are summarised in Table 1. Digestion with estEll (results not shown) indicated that all samples from Queensland and most samples from the Northern Territory were identical to TBB and SPLL- V4, which share the same pattern using this enzyme. However, the two samples from Katherine (K7-YC and K8-YC) that produced the unique fragment pattern with AZul and RsaI gave the SPLL fragment pattern with this enzyme. Australasian Plant Pathology Vol. 28 (2) 1999

Sequence analysis Sequence comparisons between the papaya sample K7-YC from Katherine and SPLL-V4 showed that the spacer sequence of K7-YC which produced the unique RFLP pattern when digested with AluI and RsaI had two insertions and two mismatches when compared to SPLL-V4. The spacer sequence of SPLL-V4 is the same as TBB except for a deletion very early in the spacer region (data not shown).Anotherphytoplasma from papaya (K6-Y C) was sequenced and found to have a deletion at the same position as SPLL-V4 even though the RFLP results indicated this sample to be

of the TBB type. This observation is reflected in the dendrogram (Figure 3) in which K6-YC is grouped with SUNHEMand SPLL-V4. This suggeststhat the differences between TBB and SPLL-V4 observed with RFLP analysis arise from changes in the 16S rRNA gene rather than the spacer region. All spacer regions contained the tRNN'e gene. The similarityof the K7-YC and SPLL-V4phytoplasma sequences to those of samples collected in Asiaand Australiais depictedwithinthe dendrogram (Figure 3). Results showed that K7-YC which produced the unique fragment pattern (see Figures 1C

Table 1 Detection of phytoplasmas in papayas with yellow crinkle (YC) or mosaic (M) by PCR and fluorescence microscopy Sample designation" Rl-YC R2-M R3-M R4-M R5-YC R6-M R7-M R8-YC R9-YC RIO-YC RII-YC Cl-YC C2-YC C3-YC C4-YC C5-YC C6-YC C7-YC KI-YC K2-YC K3-YC K4-YCD K5-YCD K6-YCD K7-YCD K8-YCD

PCR result" undiluted DNA 1:10 dilution

DAPI result

TBB SPLL-V4 TBB TBB TBB

+ + + + + +

+ nt + +

RFLP result"

+ + + +

TBB TBB TBB TBB

+

+ + nt + nt nt nt

+ + + + + + +

TBB TBB TBB TBB TBB TBB TBB

+ + +

nt nt nt

+ + +

SPLL-V4 SPLL-V4 TBB SPLL-V4 SPLL-V4 TBB CWB CWB

+ + +

APapaya samples were collected from Rockhampton, Queensland (R I-RII), Caboolture, Queensland (C I-C7) and Katherine, Northern Territory (Kl-K8). M = mosaic; YC = yellow crinkle. B+/_ = positive/negative PCR result; nt = not tested. cRFLP results where SPLL-V4, TBB and CWB refer to sweet potato little leaf variant V4, tomato big bud, and cactus witches' broom, respectively. DDNA and PCR product of samples K4-YC to K8-YC were provided by Richard Davis (Northern Territory University, Darwin, Northern Territory, Australia). Australasian Plant Pathology Vol. 28 (2) 1999

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Figure I AZul restriction digests ofPlIm23sr PCR products. Band sizes in base pairs (bp) indicated to left. A PYC samples collected from Caboolture, Queensland. Lane I = molecular weight marker. Lanes 2 to 8 = samples CI-YC to C7-YC, respectively. Lanes 9 to II = SPLL- V4, SPLL, TBB, respectively. B PYC and PM samples from Rockhampton, Queensland. Lane I = molecular weight marker. Lanes 2 to 6 = samples RI-YC to R5-YC, respectively, 7 to 10 = R7-YC to RI 0-YC, respectively. Lanes 11 to 13 = reference phytoplasmas as in Figure IA. C PYC samples collected from Katherine, Northern Territory. Lane I = molecular weight marker. Lanes 2 to 9 = K 1-YC to K8- YC. Lanes 10 to 12 = reference phytoplasmas as per Figure IA. 112

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Figure 2 RsaI restriction digests ofPl/m23sr PCR products. Band sizes in base pairs (bp) indicated to left. A PYC samples collected from Caboolture, Queensland. Lane I = molecular weight marker. Lanes 2 to 8 = samples CI-YC to C7-YC. Lanes 9 to II = reference phytoplasmas as per Figure IA. B PYC and PM samples from Rockhampton, Queensland. Lane I = molecular weight marker. Lanes 2 to 10 = samples RI-YC to R5-YC, R7-YC to RI 0-YC. Lanes 11 to 13 = reference phytoplasmas as per Figure IA. C PYC samples collected from Katherine, Northern Territory. Lane I = molecular weight marker. Lanes 2 to 9 = samples K 1-YC to K8-YC. Lanes 10 to 12 = reference phytoplasmas as in Figure IA. Australasian Plant Pathology Vol. 28 (2) 1999

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F igure 3 Dendro gram s howi ng th e ge neti c re lationship s of 12 phytopl asmas from As ia and Australia based on spacer region sequence analysis. CWB = cac tu s witche s' bro om from Indonesia (Y I4646); K7- YC = papaya yellow crinkle sample collected fr om Katherine , Northern Ter ritory (YI8129); SPLL-V4 = sweet potato little leaf in periwinkle from Darwin, Northern Territory (YI8130); SUNHEM = crotal aria witches ' broom from Tha iland, and PPWB = pigeon pea witches' broom from China (C.D. Smart, Department ofPlant Pathol o gy, C orn e ll Univ er si ty , N Y, 148 53) ; SESPHY = sesame ph yllody fro m Th a il and (X83431); PYC = papaya yellow crinkle from Rockhampton, Que en sland (Y0 8 174); PM = papaya mosaic from Rockhampton, Queensland (Y08175); TBB = tomato big bud from Darwin, Northern Territory (Y 0 8 173) ; FABAPHY = fab a bean phyllody from the Sudan (X83432); SPLL = sweet potato little leaf from Darwin, Northern Territory. PileUp parameters were a gap weight of 5.0, and a gap length weight of 0.5.

Australasian Plant Pathology Vol. 28 (2) 1999

with PM. A lthough only a limited number of PM samples was available for analysis, no specific correlation bet ween phytoplasma typ e and either disease could be established using RFLP analysis of the ribosomal DNA . It is possible that the different symptoms of PYC and PM are caused by another pathogen, or that the phytoplasmas are indeed different but the mol ecular tools used were not sufficiently sens itiv e to dete ct these differences (Gibb et al. 1996b; White et at. 1997) . This study showed that there can be genetic variability within phytoplasma populations assoc iated with the one pap aya disease. The PYC popul ation from Katherine was found to contain phytoplasm as from three distinct RFLP groups. Two of these, the SPLL-V4 and TBB types are common throughout Australia (Davis et al. 1997) and their presence was not unexpected. However, the third group, which was identified by sequence analysis to be similar to CWB from Indonesia , had not pre viou sly been detected in Australia. The differ ence in the degree ofvariability within phytoplasma populations from the Northern Territory and Queen sland could be a reflection of climate differences, vector biolo gy or specificity, discrepancies in samplin g technique, or perhaps the dominance of the TBB type over the other phytop lasma types (Gibb et al. 1996a). Sequ ence analysis of the spacer re gions from Asian and Australian phytoplasmas indicated a high degree of relatedness . Thi s technique also con firmed that the new phytoplasma was similar to cactus witches' broom from Indonesi a. However, thi s compariso n is based on a relative ly sma ll number ofnucleotides (240 bp) which can result in excessi ve branch shifts in the dendrogram. The significance ofthe changes in the conserved 16s rRNA coding regi on of the di fferent phytoplasmas, as indicated by the slight RFLP pattern changes, is unknown. Until the significance of change s in such a conserved region is determin ed each rRNA operon RFLP type is recognised as a distinct phytoplasma. The polymorph isms observed in this study were found to arise from changes in the size s of the result ing fragments due to insertions between sites rather than the deletion or creation of restriction sites themselves, since the number of bands remained conserved. This is expected since the P l/m23sr PCR produc t contains the conserved sequence s of the 16S and 23S rRNA genes and the tRNNle co ding region that occu rs w ithin the intergenic spacer (Gundersen et at. 1994). However, the reference phytoplasmas TBB and SPLL -V4 113

used for this study cause different symptoms in periwinkle, which implies that apparently minor changes in the 16S rRNA gene can be indicative of more extensive differences elsewhere in the genome resulting in different symptom types. This being the case, the RFLP differences observed in the papaya samples collected during this study could possibly be correlated with different symptoms in their common host papaya, but this was not observed. Reasons for this could be that there is no relationship between these RFLP types and symptoms, the range ofsymptoms which can be expressed by this host is limited, or subtle differences in symptoms existed, but were not observed. In either case the implications of changes in the 16S rRNA region must be further studied before the importance of the various RFLP groups observed in this study can be determined.

References Ahrens, U. and Seemuller, E. (1992) - Detection of DNA of plant pathogenic mycoplasmalike organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82: 828-832. Davis, R.I., Schneider, B. and Gibb, K.S. (1997) - Detection and differentiation of phytoplasmas in Australia. Australian Journal ofAgricultural Research 48: 535-544. Deng, S. and Hiruki, C. (J 991) - Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. Journal ofMicrobiological Methods 14: 53-61. Doyle, J.J. and Doyle, lL. (1990) - Isolation of plant DNA from fresh tissue. Focus (Life Technologies Inc.) 12: 13-15. Gibb, K.S" Davis, R. and Constable, F. (1996a) - Tomato big bud phytoplasma - A widespread and' successful' phytoplasma in Australia. 10M Letters 4: 280. Gibb, K.S., Persley, D.M., Schneider, B. and Thomas, lE. (J 996b) - Phytoplasmas associated with papaya disease in Australia. Plant Disease 80: J74- J78. Gowanlock, D.H., Greber, R.S., Behncken, G.M. and Finlay, J. (J 976) - Electron microscopy of

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mycoplasma-like bodies in several Queensland crop species. Australasian Plant Pathology Society Newsletter 5 (Suppl.): Abstract: 223. Gundersen, D.E., Lee, I.-M., Rehner, S.A, Davis, R.E. and Kingsbury, D.T. (1994) - Phylogeny of mycoplasmalike organisms (phytoplasmas): a basis for their classification. Journal ofBacteriology 176: 5244-5254. Liu, B., White, D.T., Walsh, K.B. and Scott, P.T. (1996) - Detection of phytoplasmas in dieback, yellow crinkle, and mosaic diseases of papaya using polymerase chain reaction techniques. Australian Journal of Agricultural Research 47: 387-394. Padovan, AC., Gibb, K.S., Bertaccini, A, Vibio, M., Bonfiglioli, R.E., Magarey, P.A. and Sears, B.B. (J 995) - Molecular detection of the Australian grapevine yellows phytoplasma and comparison with grapevine yellows phytoplasmas from Italy. Australian Journal ofGrape and Wine Research 1: 25-31. Peterson, R.A., Coates, L.M. and Persley, D.M. (1993) - Papaya diseases. In: Diseases of Fruit Crops (Ed D.M. Persley), pp. 70-76. Department of Primary Industries, Brisbane. Sambrook, L, Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, Second Edition. Cold Springs Harbor Laboratory Press, New York, USA Schneider, B., Seernuller, E., Smart, C.D. and Kirkpatrick, B.C. (1995) - Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In: Molecular and Diagnostic Procedures in Mycoplasmology. (Eds S. Razht and J.G. Tully), pp.369-380. Academic Press, San Diego. Seemliller, E. (1976) - Investigations to demonstrate mycoplasmalike organisms in diseased plants by fluorescence microscopy. Acta Horticulturae 67: 109112. White, n.i., Billington, S.1., Walsh, K.B. and Scott, P.T. (1997) - DNA sequence analysis supports the association of phytoplasmas with papaya (Carica papaya) dieback, yellow crinkle and mosaic. Australasian Plant Pathology 26: 28-36.

Manuscript received 28 April 1998, accepted 5 November 1999.

Australasian Plant Pathology Vol. 28 (2) 1999