First report of ‘Candidatus Phytoplasma mali’ in Prunus avium, P. armeniaca and P. domestica

N. Mehle¹*, J. Brzin¹, J. Boben¹, M. Hren¹, J. Frank¹, N. Petrovič¹, K. Gruden¹, T. Dreo¹, I. Žežlina², G Seljak² and M. Ravnikar¹

¹ National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia
² Institute of agriculture and forestry Nova Gorica, Pri Hrastu 18, 5000 Nova Gorica, Slovenia

*natasa.mehle@nib.si

Accepted for publication 24/11/06

Apple proliferation (AP) phytoplasma (‘Candidatus Phytoplasma mali’; Seemüller & Schneider, 2004) is the causal agent of a serious proliferation disease of apple (Malus domestica).  It has also been found in hazelnut (Corylus spp.) (Marcone et al., 1996), pear (Pyrus communis) and Japanese plum (Prunus salicina).  In this report, AP phytoplasma was found in cherry (Prunus avium), apricot (P. armeniaca) and plum (P. domestica).

Figure 1: The wilting of cherry	Figure 2: The dying of cherry

In cherry, wilting (Fig. 1), dying (Fig. 2), floral (Fig. 3) and phloem necrosis (Fig. 4 & 5) were first observed in 2004 in south western Slovenia.  No phytoplasmas could be isolated from the leaves or phloem from 40 trees, but were detected in roots of 3 symptomatic and 1 asymptomatic tree using DAPI and EM.  These were subsequently identified as AP by PCR, nested PCR, RFLP and sequencing.  AP phytoplasma was also identified in 2 out of 29 apricot trees expressing stem necrosis and leaf wilting, and in 1 out of 34 plum trees expressing late blooming.

Figure 3: Floral necrosis on cherry	Figure 4: The necrosis on cross section of branches of cherry

Total DNA was extracted from the roots (cherry) or roots and shoots (apricot and plum) using a modified CTAB method or the Plant DNeasy mini kit (Qiagen GmbH, Hilden).  PCR was performed using universal phytoplasma rDNA primers P1/P7 (Seemüller et al., 1996), followed by a nested PCR using the AP group specific primers f01/r01 (Lorenz et al., 1995).  Controls included DNA extracts from healthy plants and AP-infected Catharanthus roseus and apple.  RFLP analysis of nested PCR products was performed using restriction enzymes BsaAI and SspI.  All positive samples produced a restriction profile identical to the AP-infected apple.  The f01/r01 PCR products were cloned into pGEM-T vector and sequenced (GenBank Accession No. EF025917-20).  BLAST analysis of 1018 bp sequences showed the highest identity (greater than 99.6%) with the 'Ca. Phytoplasma mali' (GenBank Accession No. AF248958.1).

Figure 5: The necrosis on cross section of trunk of cherry

To our knowledge, this is the first report of AP phytoplasma infecting cherry, apricot or plum (P. domestica). Although the AP-infected cherries in the present study tested negative for other pathogenic bacteria, further experiments are needed to verify whether the AP phytoplasma caused the observed symptoms.

Acknowledgements

Thanks to M. Tušek Žnidarič (NIB, Slovenia) and P. Ermacora (Univ. of Udine, Italy) for helping with the phytoplasma identification.

References

Lorenz KHB, Schneider U, Ahrens E, Seemüller E, 1995. Detection of the Apple Proliferation and Pear Decline Phytoplasmas by PCR amplification of ribosomal and non ribosomal DNA. Phytopathology 85, 771-776.

Marcone C, Ragozzino A, Seemüller E, 1996. Association of phytoplasmas with the decline of European hazel in southern Italy. Plant Pathology 45, 857-863.

Seemüller E, Kison H, Lorenz KHB, Schneider U, Marcone C, Smart CD, Kirkpatrick BC, 1998. Detection and identification of fruit tree phytoplasmas by PCR amplification of ribosomal and nonribosomal DNA. In: Manceau C, Spak J, eds. New technologies to improve phytodiagnosis: Advances in the detection of plant pathogens by polymerase chain reaction. Luxembourg: Office of the Official Publications of the European Community, 56-66.

Seemüller E, Schneider B, 2004. 'Candidatus Phytoplasma mali', 'Candidatus Phytoplasma pyri' and 'Candidatus phytoplasma prunorum', the causal agents of apple proliferation, pear decline and European stone fruit yellows, respectively. International Journal of Systematic & Evolutionary Microbiology 54, 1217-1226.

The British Society for Plant Pathology