Improved detection methods for fruit tree phytoplasmas

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.

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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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Correspondence to Maria Heinrich.

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Heinrich, M., Botti, S., Caprara, L. et al. Improved detection methods for fruit tree phytoplasmas. Plant Mol Biol Rep 19, 169–179 (2001). https://doi.org/10.1007/BF02772160

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Key words

  • apple
  • apricot
  • fruit trees
  • in vitro cultures
  • pathogen detection
  • phytoplasmas