Serological detection of viruses included in certification protocols for stone fruits Boscia D., Myrta A. in

Boscia D., Myrta A.

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 171-190

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--- To cite th is article / Pou r citer cet article

--- Boscia D ., Myrta A. Serological detection of viru ses in clu ded in certification protocols for ston e fru its. 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. 171-190 (Options Méditerranéennes : Série B. Etudes et Recherches; n. 19)

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di Studio del sui e le delle

Arben

of and Food

-

methods. The focuses on and techniques

and affecting stone

the also discussed.

words: stone plant plant

-

techniques sérologiques, en passant en revue les Trichovirus, les les les

signalés sur les essences h noyaux. On discute également les pyoblèmes qui inerferent avec la fiabilité des résultats de

essences à sérologie, des végétaux, certification des plantes.

-

technique detection, and based on the use of antibodies, of the immunoglobulin type, in animals and capable of specific binding to

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

172 Boscia,

antigens. was of in identificating and classifying

in the case of woody plants, they lacked the sensitivity diagnosis. A in sensitivity was achieved with the development of

imunoenzymatic techniques, (enzyme linked assay), which

employs antibodies conjugated to an enzyme, to amplify and signal the of amounts of antigens.

The potential application of the was limited which specific antibodies Consequently only known not diseases of unknown

can be detected by the antigenic in the

cannot be detected by this means.

- Serological methods

1.

is a diagnostic techniques utilised identlfying plant of specific antigens in infected sap is detected that develops because of the of an enzyme (alkaline phosphatase

conjugated to antibodies in the of an

sphate l),

(double antibody sandwich) is a Schematically, antigens

antibodies coating the of wells,

and, antibodies. Finally, the addition of the

induces a in the of the antigen-enzyme antibody-

conjugate complex.

The success adopted in due to the

advantages that this technique in with and Joseph,

O sensitivity detecting small amounts of antigen of 1-10 n g / d ;

O speed of

-

scale of - samples can be handled, individually

in

ct use with plant and o

o suitability both intact and of

Serological detection of viruses included in certification protocols for stone fluits

o possibility of obtaining quantitative

o possibility of automation and of tests by the and availability of

o low cost and long shelf life of

o basic equipment and supplies;

o economical technique.

has been applied to of stone since its into plant

in 1976. The was with and plum pox

of and filamentous et al.,

1976; et al., 1976). The technique was applied to the of the

which available.

Up to now, almost 30 as actual putative agents of diseases affecting

stone that in still 16

(Tab. 1) which tests have to be done the of plant

in the These include of (ACLSV);

and

ands (LCV)

and,

to

and, consequently, induce the of and

of sensitivity that, in some cases, of low diagnostic

with in the choice of sampled

tissue and the season samples collected, of in to the

as and the evaluation of the status of plants.

1.1. ( m b s )

the absolute of that have and

causal agents of was using polyclonal and

antibodies have been

stone et al., 1982a, b; 1984; and 1989; et al.,

et al., 199810; et al., was the possibility to

identify and between with

antibodies et al., et

d.,

et al., advantages of monoclonal antibodies (e.g. specificity,

unlimited of utilisation of

174 D. Boscia,

mix infected these should not be used exclusively. The this is because of its and some false negatives

and to a high level of

among isolates, can not be safely detected by single monoclonal antibodies, unless cocktails used et al., 199813).

1.2. Artificial polyvalent antisera

To limitations of specific antibodies, polyclonal to may be mixed (polyvalent et al., 1979; Uyemoto, 1980).

Thus, will detected simultaneously by using polyvalent et

al., 1983), and its sensitivity was not if conditions the

detection of the (James, 1997).

2. Other serological techniques alternative to

2.1. Assay

has the same sensitivity and need equipment. on the use

of in place of plates used and eliminate the

need plate et al., et al., 1993).

2.2. Tissue blot immunoassay

While it may not always the same sensitivity as and tissue is

(sample and used eliminated) and, as

it can be with little equipment. addition, tissue can data on localisation within plant et al., 1993; et al., 1995).

- Stone fruit viruses

1. Trichoviruses

1.1.

This is of medium and detectable by

imuno-tissue et al., has

been used ACLSV detection et al., 1981; and detecting ACLSV by with the of its isolates, difficulties

stemming low and low To this

Serological detection viruses included in certijcnfion protocols stotzefvuits 175

difficulty, Flegg and (1979) modified the technique by incubating the enzyme conjugate with the sap instead of adding it sequentially. The modified of the

technique detecting ACLSV, (Flegg and was used

et al., et al. (1980) failed

to detect ACLSV in using the same modified

obtained with in the of stabilising agents and nicotine to plant tannins.

(1980, 1982), the of ACLSV in apple

beginning in and its maximum and June. ACLSV was detected by

in using buds et al. (1980)

detected ACLSV field conditions in using

in the of

plant to test petals the detection of ACLSV

in et al. (1985) suggested tissue. and (1986)

an of ACLSV, with leaves at the base of the

than apical ones. They found that two wood was the most

tissue in the season.

Although ACLSV has a high of its antigenic

stable and polyclonal detect all known of the Any

in antigenic of had not posed any and does

not polyvalence of polyclonal the ACLSV

the and use of monoclonal antibodies and 1990;

et al., 1997).

2. Potyvirus

2.1.

virus

agent of infects many cultivated and wild species and is one of

the most disease of symptoms develop in

and was found in sweet and et al., 1994;

et al., distinct have with

in and epidemiological The

include and

and et al., et al., endemic in

in peach

lack the site in et al., et al. (1995) had

designed and was also

176 Boscia,

in coat subunits mobilities in (Adamolle,

1993; and was common in and plum and

in peach. et al. (1994, 1995) identified the by enzymatic digestion and by was isolated in Egypt. This isolate showed high level of in the nucleotide and amino acid sequences to

isolates. et al. (1998) designed based on Nib

and isolates identified

(Nemchinov and 1996; et al., 1998), and with

specific to (Nemchinov et al., 1996), and (Nemchinov and 1997).

has good immunogenic and detection by (using antibodies) had been used

et al., 1995).

tests Nyujtó et al. (1985) obtained amplified sensitivity by using avidin- biotin The method was successfully used testing and plum seeds

the (Németh and

addition to polyclonal antibodies, now being used

identification of fact, one antibody was specific a

common antigenic on all isolates et al., 1994). To

idenbfy isolates to a which

antigenic e.g. et al.,

et al., et al., 1998b), and

et al., 1998) available.

in leaves was highest in and June. was

in than in ones (Németh and was detected in

and et al., et al., 1986;

et al., 1986; Adams et al., 1998). This assessment was influenced by the host species and used et al. (1986) found that assays of peach

using of twigs and in young shoot leaves.

On was detected in and young shoots, and in late in

leaves.

testing it must be that is unevenly in infected and

so infected and can be found not only within the same leaf but also within

the may 1977;

(1983) detected in of symptoms in 9 to

analysis of an adult with the

uneven of the of 700 samples the

Serological detech’on of viruses included in certification protocols stone fruits

negatives. 15% negative when the same collections tested by

suggesting of low of

et al., 1988). To minimise sampling we suggest collecting

this is obviously a as it means taking and analysing

samples indicate that even a sensitive method like

can not

tissue was also used in host studies and

1994; et al., can be utilised also (Noel et al., 1978;

et al., 1981; et al., 1988).

3.

detected by has been noted et al., 1998b; Fulton,

1968) that is a with and

3.1. Apple mosaic virus

( A p m

was detected by et al., et al., 1976; et al.,

et al., 1979; et al., 1979; and

and

detection can be done the season in individual samples of young

with newly buds, and in June

and detection was easiest to mid-

June (Fuchs, 1980).

in between tissues of plant

As with ACLSV, the petals

in

Studies made with monoclonal antibodies in two and

1991; et al., 1998b) indicated that the of the population highly stable and, consequently, single suitable detection.

3.2. virus

was and

and detection was in

avizm seeds and is used in testing of and

aviunt, mahaleb, cerasifera, and persica and Aichele, 1984a).

178 Boscia, A.

was developed et al. (1986) and

diagnosis in seeds. The test a quick assessment of seed lots, issue of with monoclonal antibodies was applied to study

seed in mahaleb et al., 1998a).

plum and sweet was detected the whole vegetation in young

in newly buds and

isolates by et al. (199813) identified

36 in 128 isolates species. Consequently,

if monoclonal antibodies used assays, would be advisable to

cocktails of to of false negatives.

3.3. necrotic ringspot virus

was widely used the detection in tissues collected in the vegetation et al., 1977; et al.,

and Aichele, 1984 a, b; and method was also used in assays of seedlings of cerasifera, and persica and Aichele, 1984a). A

specific study out and that can be

detected in the whole vegetation in young leaves in newly

buds. The sensitivity was when an amplification of the

enzyme was applied tissue effectively detected

as well et al., 1995).

1973; et al., 1987), and must be given when using of its high specificity, false negative may be

et al. (199813) had identified 17 among 38 isolates tested.

Consequently, as done and when using the adoption of cocktails must be the past, high specificity of polyclonal was also it led to identification of one isolate, initially classified as but as

4. Nepoviruses

in good immunogenics and can be detected

et al., 1976; et al., et al., 1976;

1977, Gonsalves, et al., 1980; and et al.,

1984; and etal., et al., 1984; and

Gonsalves, 1986).

detection of viruses included in certification vyotocols for stone fruits 179

was developed et assays of

seeds. This test a quick assessment of seed lots the issue of

have been et al., 1982). Antigenic lead to a specificity of

detection of tomato the

was in some e f al. (1980) that was

detected in apple in leaf- and of and

in et al. (1984) showed, in apple that this was in leaves, slightly less in the and the

the end of the season, the of the assays and

Gonsalves (1986) found the in peach to be but mostly

at and below the soil line.

5. Closteroviruses

defined by on sensitive Sam),

was in with and

developed et a1.,1996).

the association of this to the

basically to the the of the and the

of symptoms.

6. Foveaviruses

A the detection of mottle was

developed (Zhang et al., 1998). No available.

7. Tombusviruses

mosaic was unevenly within

was to the tissues in the plant tested (leaves,

young twig-tips, showing no symptoms of the disease often negative in test. indexing latent infections with

means of not possible at the and 1996).

180 D. Boscia, A.

problems

1. Sampling

The of the sampled tissues has influence on the in

tannins and oxydative substances, should be avoided. samples, the

often consists of budwood and may be

done in ways: i) bioassays by bud and onto testing; ii)

to assay tissues; and iii) analysis of the

Although in may be low, based

assays can be done with a amount of success.

2. Uneven distribution of the. pathogen in the infected plant

This phenomenon was with many infecting stone Not

only when was localised a point of infection, but also in classical systemic

infections, when the was unevenly in

in the plant. This happens in woody plants but

apply to as well.

3. Grouping of samples

is the method testing of of samples test

of samples. to and the sampling date influences

also the efficiency of was detected in 1/40

(infected/total leaves) leaves in and and plum leaves up to July, and was detected in 1/20 leaves until July and in apple and plum leaves to

was detected in 1/20 apple and plum leaves to July.

4. The variability of pathogens

The may be highly specific, conjugate a given

unable to So, with distantly

some also ones, go undetected 1978;

et al., 1980). The high of selectivity may be an advantage in epidemiological studies specific must be distinguished. in diagnostic and extensive testing all the of a both known and unknown, must be detected, it is

Serological detection of viruses included in certilfication protocols stonefluits 181

As by van and and Lommel et al. (1982),

specificity does not a with the (TAS) systems in which,

instead of labelled with the enzyme. ^~

Although ACLSV has a high of at the symptom level, its antigenic

stable and polyclonal detected all known and

(1986) on the existence of of ACLSV. study

involving eight and 29 and (1989), that the

antigenic et al.

(1995) in the analysis of coat genes of ACLSV isolates.

5. Serological cross-reactions

have been and of stone

between of the same taxon even

et al. (1994; 1996) with a polyclonal

antibody and isolates, (1991)

with isolates.

between and have been (Fulton, 1968;

et al., using have not

shown any at least not with and

1991; et al., 199813).

l

V - Conclusions

tests useful as detection methods in and

in due to low cost and

we must avoid due to the

choice in the time of and method of sampling, quality of and conduction

methods may not satisfy all assessment of

the plants (biological assay still play an in a

of detection and adaptability and assays of

sample as sensitivity, should now

be taken in it involve small of

-

polyclonaux spécifiques de non de la bioécologie de deux

épidémiques en 153 pp.

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in hosts by enzyme- linked assay. Acta Academiae Scientianlm Hungaricae, 15: 329-332.

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Serological detection of viruses included certification protocols stone fruits 189

LChV

ACLSV

Tab. indicated in the to assess the status

of stone (Anonymous, 1992)

Taxonomic Availability of mottle Foveavirtls

Little Closterovirus

Apple mosaic

leaf leaf latent

latent Tomato black Tomato

pox

mosaic Tornbusvirus

Apple leaf spot Trichovinls

No No Y es Y es Yes Yes Y es Y es Y es Yes Y es Y es Y es Yes No Yes

190 D. Boscia,