Plant Molecular Biology Reporter

, Volume 19, Issue 2, pp 169–179 | Cite as

Improved detection methods for fruit tree phytoplasmas

  • Maria Heinrich
  • Simona Botti
  • Licia Caprara
  • Wolfgang Arthofer
  • Sabine Strommer
  • Veronika Hanzer
  • Hermann Katinger
  • Assunta Bertaccini
  • Margit Laimer Da Câmara Machado
Protocols

Abstract

Phytoplasmas infecting fruit trees are considered quarantine organisms in Europe and North America. Detection often is hampered by their extremely irregular distribution in host plants. A sensitive, specific and quick diagnostic test would be highly desirable for routine detection, mainly to avoid using infected planting material. PCR methods require tedious preparation of DNA; also, the available primers are highly specific and exhibit some homology to chloroplast and plastid DNA. To address these problems, we compared several DNA preparation protocols for purity of DNA, cost and time required. We also developed new primers using rDNA sequence information from an Austrian isolate of European Stone Fruit Yellows (ESFY). These primers operate at high annealing temperatures and, thus, increase the specificity and decrease the risk of false positives. The primers could reliably detect the European phytoplasmas (AP, ESFY and PD) within a collection of isolates maintained in micropropagated periwinkle. Thus, they are suitable as general primers for phytoplasma detection. The primers also can be used for strain identification by direct PCR followed by RFLP analysis as demonstrated with micropropagated fruit tree material. Finally, an IC-PCR method that uses the primers for AP detection was found very sensitive and suitable for large-scale testing of apple materialin vivo andin vitro.

Key words

apple apricot fruit trees in vitro cultures pathogen detection phytoplasmas 

Abbreviations

ACLSV

apple chlorotic leafspot virus

A-AY

American aster yellows

AP

apple proliferation

ASGV

apple stem grooving virus

ASHY

ash yellows

ASPV

apple stem pitting virus

AT

apple proliferation strain

BVK

phytoplasma fromPsammotettix cephalotes

CHRY

chrysanthemum yellows

DAS-ELISA

double antibody sandwich-enzyme linked immunosorbent assay

ESFY

European stone fruit yellows

EY-C

elm yellows

FBPSA

faba bean phyllody

GVX

Green Valley X disease

GSFY

German stone fruit yellows

IC-PCR

immuno capture-polymerase chain reaction

KVM

clover phyllody

LNS

plum leptonecrosis

LUM

Lucerne virescence

MOL

Molière disease

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Copyright information

© International Society for Plant Molecular Biology 2001

Authors and Affiliations

  • Maria Heinrich
    • 1
  • Simona Botti
    • 2
  • Licia Caprara
    • 2
  • Wolfgang Arthofer
    • 1
  • Sabine Strommer
    • 1
  • Veronika Hanzer
    • 1
  • Hermann Katinger
    • 1
  • Assunta Bertaccini
    • 2
  • Margit Laimer Da Câmara Machado
    • 1
  1. 1.Plant Biotechnology Unit, Institute of Applied MicrobiologyUniversity for Agricultural SciencesViennaAustria
  2. 2.DiSTA-Patologia VegetaleUniversità degli Studi di BolognaBolognaItaly

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