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FAIR 3889

Virus diseases

Phytoplasma diseases

Pathogen collection

Pathogen detection

ACLSV
ApMV
ASGV
ASPV
PPV
PDV
PNRSV
ArMV
ToRSV
RpRSV
SLRSV
GFLV
GLRaV-1
GLRaV-3
LChV
CMLV
CRMV
CNRMV
CGRMV
ChTLV
CVA
AP
ESFY
PD

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Pathogen detection


 Different methods for the detection of plant pathogens have been developed and are applied, depending on the pathogen to be detected, the time frame, equipment and financial ressources available. These aspects are important for the choice of a test system, when to be applied for traded budwood.

Internationally approved and recognized detection systems including serological and molecular laboratory assays and indicator hosts in greenhouse and field indexing were updated regularly by the ISHS International Working Group on Fruit Tree Viruses in occasion of their meetings 1991 in Vienna (Acta Hort. 309: 407-418, 1992), 1997 in Bethesda (Acta Hort. 472: 761-783, 1998) and 2000 in Canterbury (Acta Hort. in press).

An overview of these methods for pome fruit and stone fruit pathogens is given in the tables at the bottom of this page.


Viruses

 Viruses can be detected by field and greenhouse indexing, serology, optical methods like electron mircoscopy, molecular hybridization, Nucleic Acid Sequence Based Amplification (NASBA) and PCR amplification. The ISHS ´Working Group on Virus Diseases of Fruit Trees´ recommends serological indexing (ELISA) since many years. Since the Bethesda meeting in 1997 major emphasis is layed on the application of PCR based detection systems, due to their increased sensitivity and velocity.
In the frame of FAIR 3889 improved protocols both for RNA preparation but also for specific and broad range detection of fruit tree viruses have been established.

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Phytoplasmas

 Phytoplasmas may be detected by orchard and nursery inspections due to typical morphological anomalies. ESFY can be reliably detected by indexing on GF 305 seedlings in the greenhouse with an incubation period of approximately 4 months. In the sieve tubes of petioles, bark and roots phytoplasmas can be detected by DAPI staining.
In the frame of FAIR 3889 PCR was optimized as rapid and reliable diagnostic tool. The PCR detection of phytoplasmas relies on the quality of the DNA template for amplification. Therefore different protocols for DNA preparation are recommended. Plate Capture PCR or IC-PCR avoids an initial preparation step by use of antisera.

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Indexing

 Indexing is based on the ability of special indicator plants to develop typical disease symptoms after infection with a certain virus. The cultivar to be tested is grafted to the indicator or plant sap is trasmitted mechanically. After a given incubation time the presence of visible symptoms is determined.

The following tables give an overview of approved indexing methods and the viruses that may be detected on indicator plants.

Further information about indexing is available on the homepage of the Washington State University.

TypeIndicatorDuration Detectable Viruses
Herbaceous Greenhouse Indexing Chenopodium quinoa20 days ACLSV, ASGV, Nepoviruses
Cucumis sativus20 days ApMV, PDV, PNRSV
Woody Greenhouse Indexing Malus platycarpa8 weeks ACLSV
Malus pumila ´Virginia Crab´24 weeks ASGV, ASPV
Malus pumila R 12740 7A4 weeks ACLSV
Malus pumila spy 22712 weeks ASPV
Cydonia oblonga C 7/15 weeks ACLSV
Prunus persica GF 3058 weeks ACLSV, PPV, PDV, PNRSV, SLRSV, ESFY
Prunus tomentosa12 weeks ACLSV, PPV, PDV, PNRSV
Prunus serrulata ´Shirofugen´8 weeks PDV, PNRSV
Field indexing Malus platycarpa2 years ACLSV
Pyronia veitchii2 years ASPV
Malus pumila ´Virginia crab´3 years ASGV, ASPV
Malus pumila R 12740 7A2 years ACLSV
Malus pumila spy 2272 years ASPV
Malus pumila ´Lord Lambourne´3 years ApMV, rubbery wood, flat limb, chat fruit
Malus pumila ´Gravensteiner´3 years flat limb
Malus pumila ´Golden Delicious´2 years ApMV, AP
Prunus serrulata ´Shirofugen´6 weeks -
2 years		 PDV, PNRSV
Prunus serrulata ´Kwanzan´2 years CGRMV
Prunus avium ´Bing´3 years CRLV, CTLV, SLRSV, ArMV, CNRSM, CRMV
Prunus avium ´Sam´3 years LChV
Prunus avium Canindex I3 years CNRMV
Prunus persica GF 3054 years ACLSV, PPV, PDV, PNRSV, ESFY

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Serology

 The ELISA (Enzyme Linked Immunosorbant Assay) test has been developed for the detection of plant viruses almost 30 years ago. It is used routinely for large scale testing of plants for the presence of many viruses. It is rapid, inexpensive and convenient. However, ELISA can only be applied for those viruses where specific antisera are commercially available.
It is further limited by the eneven patterns of distribution of certain pathogens in a tree, but also by climatic influence, reducing the titer below the possible level of detection.
In sanitation programmes after elimination treatments it may therefore require some time before virus replication reaches again a detectable treshold.
The use of Immuno-Tissue-Printing allows the localization of viruses in tissues and therefore the improvement of elimination strategies in vitro.

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Polymerase Chain Reaction (PCR)

 Polymerase Chain Reaction is a method for amplification of specific DNA regions, producing easily detectable amounts of DNA fragments, which are usually visualized by agarose gel electrophoresis.
Concerning pathogen detection, PCR is 100- to 1000-fold more sensitive than ELISA. Therefore it is specially suitable for the detection of infections in an initial stage, where the pathogen titer in the plant is still low.

Many descriptions of the PCR technique exist in the World Wide Web. Following you find a few links about this topic:

"What the heck is PCR" by C. Brown
"Principle of PCR" by Andy Vierstraete
Short description of PCR from the University of Califormia
Detailed PCR protocols from the University College in London


While the DNA of phytoplasmas can be used directly as a PCR template, most plant viruses contain RNA. For their detection either an RNA purification or, in case of pathogens for which antisera are available, an immunocapture (IC) followed by an RT step is required to convert the viral RNA to a DNA that can be amplified by PCR.

For pathogen detection PCR is carried out by using a range of general or specific primer pairs which represent short DNA patterns of the template DNA and flank the amplified region. Primers for several important fruit tree pathogens where developed in the frame of FAIR 3889.


PCR products can be further analyzed in RFLP by enzymatic digestion. The resulting ´fingerprints´ confirm the identity of the amplified products and may be used to identify sub-strains of a certain pathogen.

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Serological and molecular laboratory assays available for the detection of viruses and phytoplasmas in pome fruits
Pathogen
(click for detection details)		 Serological AssayMolecular AssayReference
ACLSVELISA RT-PCR
IC-RT-PCR
RT-PCR ELOSA		 6, 27, 30, 31, 41
ASGVELISA RT-PCR
IC-RT-PCR
RT-PCR ELOSA		 9, 27, 29, 30, 31, 37, 39
ASPV  RT-PCR
NASBA		 25, 29, 30, 31, 38, 40, 58
ApMVELISA RT-PCR
IC-RT-PCR
RT-PCR ELISA		 5, 56
ToRSVELISA RT-PCR
RT-PCR ELISA
Hybridization		 18, 19, 43, 56
APELISA PCR
IC-PCR		 8, 21, 35, 36, 59
PD  PCR 8, 10, 12, 35, 36, 59

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Serological and molecular laboratory assays available for the detection of viruses and phytoplasmas in stone fruits
Pathogen
(click for detection details)		 Serological AssayMolecular AssayReference
ACLSVELISA RT-PCR
IC-RT-PCR		 6, 27, 30, 31, 41
ApMVELISA RT-PCR
IC-RT-PCR
RT-PCR ELISA		 5, 56
PPVELISA RT-PCR
IC-RT-PCR		  
PDVELISA RT-PCR
IC-RT-PCR		 42
PNRSVELISA RT-PCR
RT-PCR ELISA		 5, 20, 60
ToRSVELISA RT-PCR
RT-PCR ELISA
Hybridization		 18, 19, 43, 56
ArMVELISA RT-PCR
Hybridization
NASBA		 19, 37
RpRSVELISA NASBA  
SLRSVELISA NASBA  
LChV  RT-PCR 26, 28, 46, 49, 50, 53, 62
CMLVELISA    
CRLVELISA    
CVA  RT-PCR
IC-RT-PCR		 22
TRSVELISA    
TBRVELISA    
ESFY  PCR 1, 8, 11, 13, 21, 23, 32, 35, 59

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References

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  51. Rott M. E., and Jelkmann W., 2000. Complete nucleotide sequence of cherry necrotic rusty mottle virus. Acta Hort. (in press).
  52. Rott M. E., and Jelkmann W., 2000. Development of PCR primer pairs for the characterization and detection of several related filamentous viruses of cherry. Acta Hort. (in press).
  53. Rott M. E., and Jelkmann W., 2000. Molekulare Charakterisierung eines zweiten Closterovirus assoziiert mit der Kleinfrüchtigkeit der Sübkirsche (little cherry). Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft Berlin-Dahlem (im Druck).
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last updated October 8, 2001 by Siegfried.Huss