Molecular diagnostic techniques and their potential role in stone fruit certification schemes Pallás V., Sanchez-Navarro J.A., Canizares M.C. in

Pallás V., Sanchez-Navarro J.A., Canizares M.C.

in

Di Terlizzi B. (ed.), Myrta A. (ed.), Savino V. (ed.).

Stone fruit viruses and certification in the Mediterranean countries: problems and prospects

Bari : CIHEAM

Options Méditerranéennes : Série B. Etudes et Recherches; n. 19 1998

pages 191-208

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--- Pallás V., Sanchez-Navarro J.A., Canizares M.C. Molecu lar diagn ostic tech n iqu es an d th eir poten tial role in ston e fru it certification sch emes. In : D i Terlizzi B. (ed.), Myrta A. (ed.), Savino V.

(ed.). Stone fruit viruses and certification in the Mediterranean countries: problems and prospects. Bari : CIHEAM, 1998. p. 191-208 (Options Méditerranéennes : Série B. Etudes et Recherches; n. 19) ---

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Vicente Jesús A.

Paloma

Jose F.

de

-

economic losses. At the moment, unlike and fungal diseases, no chemical exist to be applied to diseases. So, the detection by means of sensitive

diagnostic methods is the main way to them. The made in the

nucleic acid and the application of in plant

have the use of diagnostic methods based on the genomic component of

and Among them, and chain

( E " ) have lately and have been in the diagnostic field of

plant This focuses on the of these two techniques,

application to the diagnosis of stone and and potential into the

words: stone non

diagnostic methods.

-

Options Méditerranéennes, Série B / n ^O 19, - Stone fruit viruses and certification in the Mediterranean: problems andprospects

192 V. et al.

dans l'étude des acides nucléiques et l'application de la technologie d u recombinant ont permis d'utiliser des méthodes de détection basées sur la composante génomique des virus et des viuoïdes.

ces techniques, l'hybridation moléculaire et l'amplifcation de séquence ont suscité un grand intérêt et elles ont été adoptées pour le diagnostic en virologie végétale. Le présent travail illustre les bases moléculaires de ces deux techniques, leur application au diagnostic des virus et des viroïdes des essences à noynux et leur possible introduction dans des programmes de certification.

airus des essences à noynux, viroïdes, hybridation moléculaire non radioactive, méthodes de diagnostic.

-

Stone affected by a of that exlubit

biological as well as and

They belong to such as

(ACLSV), and

caused by these slight (as in the case of

ACLSV) to and Scott, 1992). Two

known to infect stone hop stunt detected as causing a

stunt disease of hops, and found in and almond (Shikata, 1990;

et al., of dapple disease in plums and peaches (Shikata, 1990).

peach latent and the causal

agent of the peach latent and in

high incidence in in of peach and

et al., 1992; et al., 1997; and et al., 1998). In

addition, has occasionally been detected in and

in et al., 1997).

At the and fungal diseases no chemicals exist that could be used

of diseases, and the detection by means

of sensitive diagnostic methods is the main way to them 1991;

on both called molecules (nucleic

acids) and functional molecules plant diagnosis have evolved in a way to the in the knowledge of these components. Until

only methods based on the component of the used in

plant detection. Among them, the used due to

use, sensitivity and automation. disadvantage of in the fact that

only 2-5% of the genetic of on the

of the coat can not be applied

to diagnosis because lack of specific-encoded

detection must on detection of the genomic

diamostic techniques ^ami their potential role in stone fruit certification schemes 193

bioassays not populations.

techniques, used on the basis of the distinct mobility .of small WAS, would

not be suitable sample The made on the nucleic

the last and the application of to

plant have using diagnostic methods based on the nucleotide sequences

of the genome component of and and

chain into the diagnostic field of plant

of these two application to the

diagnosis of stone and and potential into the

additional on

and and technologies, and

have been published.

-

Basic methodology

as a diagnostic tool in plant was used to detect

(Owens and and applied to plant et 1983; et al.,

based on specific between

and bases A-T and G=C in a stable by

the totality) of the nucleic acid sequence of the pathogen to be detected

molecule) and the The stability of the

depends on the of bonds and on both and

on the phosphate molecules of the nucleic acid

backbone maintained between the bases.

method the dot-blot technique,

of a nucleic acid solution to a solid such as and subsequent detection with

the steps of the technique (which

include the synthesis of the sample and detection)

will these steps can be found in

1993; et al., 1998).

2. Synthesis of the Probe

Use of to label nucleic acids, made the

technique and being used in an of

biotin and

194 V. e t al.

digoxigenin molecules most widely used. The biotinyl labelled nucleic acids

with efficiency by avidin taking

advantage of the exceptionally high affinity of the avidin-biotin complex. The main disadvantage of this system when sap used, the endogenous

biotin may cause false positives the of that bind

avidin biotin-binding to high widely

used molecule to the hapten digoxigenin which is

bound to and

enzymatically into nucleic acids by methods.

and most of the plant including the totality of affecting stone

have genomes. stable than

conditions can be selected in the case of

that will help to and

to detect stone

synthesised by the digoxigenin hapten into a

means of an in cloned 1A). To check the

success the yield of the the in of

the obtained in the and absence of the

must be the digoxigenin was into the the

mobility of the than that of unlabelled (Fig.

may be diluted and spotted on nylon which developed as below (Fig. 4).

detecting and affecting stone have been obtained et

al., 1993; and et al., 1995; et al.,

(Nemichov et al., 1996), CLSV et al., et al., 1995;

and et al., 1997, and et al., 1995;

et al., 1996).

3. Sample preparation

no sample conditions

analysis. Choise of depend on the being detected, the host, and the method used detecting the digoxigenin-labelled nucleic acids.

instance, when sap used, the of leaves on

the with the due to the

of the by components of the plant sap, while the light emission may not be by the of these components et al., 1993; et

al., 1998). An that of the stone assays is the

one that was applied et al., 1987) and consists of 50 mM sodium 8.3,

role in stone fiuit certification schemes 195

containing 20 mM and 2% (w/v)

at 5000 g min at 4OC, and by heating at 60°C 15 min in the of This last ‘step is

optional since it the it is

due to high of et al.,1984;

et al., 1996). methods used use

of phenol them

that of an method that avoids the use of

phenolics, obtaining plant et al., 1983), to

in et al., in the

of double and was adapted

et al., 1996; et al., outline of the method, see Fig. 2.

analysis in sample manipulation must be

to a minimum. This was achieved by using the technique that avoids

sample and o d y the of the plant

leaf) onto a nylon was to detect

by immunocytolocalization (Cassab and 1987, applied to and Guilfoyle, 1989), and then, adapted and localisation of

plant et al., 1990; Chia et al., and of stone

the nonisotopic has been applied to

and and unpublished and

et al., 1995; et al., 1996). was used

to detect ACLSV and et al., applied to

by using cotton buds and them until

and e t al., 1996).

4. and nucleic acid detection

Samples (nucleic acids) must be fixed onto a by baking 2 h at 8OoC, at 120°C 30 mni, by uv (in the last two

can be used). The last method in in the

baking methods.

The depend on such as the complexity (length and

composition of the of the the salt

and at which

half of the is the melting of the

conditions and the stability of the complexes the

specificity of high and low salt

196 V. et

of in the

and noise. plant

detection, often out at signal to

is achieved at 70-72°C in 50%

immediately The labelled

detected by an using conjugates composed of high-affinity

antibodies coupled to alkaline phosphatase washing steps, a is

obtained by subsequent addition of the and

with the chemiluminescent

- Polymerase chain reaction techniques

1. Basic methodology

The as detection method is and

sensitive. has been used in plant pathology the detection and diagnosis of pathogens as phytoplasma, fungi, and nematodes

and

and have been detailed in

this this section will the useful in the potential

application of the technique in of stone The method

utilises an enzymatic and an exponential amplification of 5).

This goal may be achieved multiple cycles of steps at

to: (i) the anneal two oligonucleotide to the

and (iii) extension by to

synthesise the sequence whose ends defined by the of

amplified can be the case of

pathogens and most plant step must be

included to copy the into to being amplified.

2. Considerations, advantages and disadvantages of

a specific and sensitive technique suitable is depended

upon the design of specific to initiate obtained

the pathogen genome. Oligonucleotide must be 18-25 (annealing) nucleotide in length, with a 50 % G+C content, no annealing ends, no

and high G+C content at the 3’ ends. may be to (to

amplify sequences of pathogens) (to between

The annealing of will affect specificity and successful

role in stonefiuit certification sclzemes 197

depend on length, its G+C content, and 20 nucleotides of up to 2°C A T and 4°C G C. With stone

was applied in detecting and

(Wetzel et al., and 1992; et al., et al.,

1995; et al., et al., et nl., 1997; et al., 1998)

the incubation the steps and

of salts and and enzyme. Size of the amplified

is also e.g. of 200-500 (no than 1000) to maximise

et al. (1997), by using two of yielding a

(200 bp) long (785 bp), have shown that was detected

in plant tissues with a low of

the the long was at Since

the of detailed to optimise

the assays must be The influence of sample and

should also be tested in a stage at the design of the diagnosis

the sensitivity of the technique allows amplification of a single nucleic acid

molecule a complex of this caution must be

taken in to minimise contamination and 'one-use' aliquots should be

of and and negative

must be included with assays

nucleic acid may be used to check and

optimise and the

those and may specialised

equipment. like those obtained with the

method (Fig. 2), may be used to suitability use.

An pointto will be the design of to

of the dilutions of samples added to positive

Stone in known to inhibit

of such test samples may be achieved by using

polyvinil which binds polyphenolic compounds.

used to eliminate in assays of (e.g. Yang, 1990; Levy et al., 1994). A method the of high-quality woody plants

that employs spin-column and the

effects of plant and polyphenolic compounds has et al., 1997).

Although unquestionable in advantages, is

such and cost of enzymes and

198 V. et nl.

i

addition, ' to false positives due to the

sensitivity and with the ease of contamination contaminated

of autoclaved

containing et al., 1992; and it

might not scale indexing. the sensitivity

make a convenient testing plants.

3. for diagnosis of viroids

was successfully applied in the detection of pome and Yang, 1990) and stone et al., 1995; et al., 1992; 1997), (Yang et al., 1992), et al., 1992; Staub et al., 1995; Wan Chow Wah and Symons, 1997), and avocado (Schell et al., the of the tissue to be analysed

the low of the made the use of and time-consuming sample

Good obtained

methods made conducted to simplify these

methods, with the use of (Levy et al.,

not solvents (Wan Chow Wah and Symons, 1997).

showed that a simple 2) et al., 1996) may be

used, coupled with to detect in et al., unpublished).

this will be useful with stone species.

designed detection, it should be noted that

of molecules (due to the high of of

compete annealing. To this oligonucleotides

with annealing

usually done at annealing no than 60°C. to maximise the detection of all sequence should be located in the well known

sequences which includes the in and the

in et al., 1996; C. and is

not to amplify complete sequence and equally good obtained

with amplified was that combinations

in yields and sensitivity of the and Symons,

1997).

- Combined techniques

and techniques not only in the component to be detected but in specificity, sensitivity, and facility of automation. the specificity and facility of automation methods combined with the sensitivity of the

and their potential role in stone f l u i t ceutification schemes 199

technique in a single assay in which initially and then

amplified by (Jansen et al., 1990; Wetzel et al., 1992; Nolasco et al., 1993). This attfactive

was 250 than

avoids using the level of of the usually in to

eliminate the compounds that methods. it

was shown that the step may be substituted by immobilisation,

not et al., 1995), and the

detection levels achieved by the than those

was the by Olmos et al. (1996) in which the simplicity of the technique (see above) was combined with the specificity and sensitivity of the technique, called

avoids the need the samples without lost of sensitivity. This technique was applied detecting and ACLSV (Olmos et al., 1996).

V - Concluding remarks and future prospects.

methods plant being continuously

on had advanced in

detecting the genomic components of plant Although and

have gained new levels of sensitivity to an acceptable level

of automation is lacking. a stone between

simplicity of automation and sensitivity must may be assayed

methods, but such as those

the could be used as well as

budwood sanitation

The simplicity and sensitivity of new methods have been sufficiently to detect most of plant at levels below economic The goal the coming

will on making these methods accessible to non

Acknowledgements

is by the de

de España and the

Union. J.A. S-N, and F.A. of

the de Educación y Spain and was of

200 V. et al.

cloned in a

4

of

, (specific

1 2 3 4

(A) Schematic diagram of the procedure used for the synthesis of a viral-specific

digoxigenin-labelled The cloned sequence is by an open

box the of an specific of T3, T7 phage

is of the sequence and then in

to the (the digoxigenin hapten is

by diamonds). (B) Agarose gel electrophoresis for the analysis of digoxigenin-labelled

probes. Lane 1, weight lane 2, plasmid lane 3,

synthesised the plasmid shown in (2) in the of non-labelled lane 4, synthesised the plasmid shown in (2) in the of (note the slight due to the of the digoxigenin hapten).

teclzniques and their potential role in stonefvuit certification schemes 201

0.5 tissue + 5 ml TE mM 0.5 10 mM ß-"E)

Sealed plastic bag

1 ml homogenate

+ 50pl 20 % 20 min 65 "C

W ⁺

0

Spn.

4

40 of

i

! . .. .--," -- i !

L_I_____ --.i

2. Schematic diagram of the non-organic sample processing procedure used for viroid detection.

The has been adapted small volumes and managing a of

samples. Aliquots of the samples in finally dotted onto Nylon fixed by uv and and developed as outlined in Fig. 4.

202 V. et al.

A _{I H}

Fig. 3. detection by of to a digoxigenin-labelled specific

Leaves leaf healthy walnut

onto Nylon Leaves

onto the (A), tightly cut with a blade, and

the section onto the analysed and developed as

outlined in Fig. 4.

role stonefluit certification schemes 203

..

Cotton of

4. Schematic diagram of the procedure used for the analysis of samples by nonradioactive molecular hibridisation. The samples on the to a specific

digoxigenin-labelled at 50-68 T in the of 50% the

is detected by immunological binding to an antibody conjugated to

phosphatase (in this the is

developped with a the conjugated enzyme, chemiluminiscent (in

this this example, samples applied onto the by

.the 'cotton-bud' technique (see text).

204 V. et al.

1 2 3 4 5 6

5. (A) Schematic diagram of the procedure used for the detection of plant viruses and

viroids. The pathogen (wavy line) is using a specific

antisense (as) oligonucleotide and then exponentially amplified multiple cycles of using specific sense (a) and antisense (as) oligonucleotides. (B) Agarose gel electrophoresis of the products obtained in the analysis of field samples for the

presence of Lane 1, weight lane 2, sample

infected peach GF-305; lanes 3 to 6, samples (samples 3 to 5 found infected with while sample 6 was

and their potential role in stone puit cerfijïcation schemes 205

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