Evaluation of Molecular and Serological Diagnostic Techniques for a Large Scale Detection of Plum Pox Virus
Plum pox virus (PPV), the agent of sharka, is the most devastating virus infecting stone fruits. The PPV control is mainly based on prevention, and its quick and reliable detection is considered crucial in this strategy. In this study DAS-ELISA and Real-time PCR were compared for evaluating their potentialities and limits for large scale surveys. Different hosts (apricot and peach), plant organs (phloem, buds, flowers, leaves and fruits) and parts of them, different periods of the year, the presence or absence of symptoms were considered for comparison. No significant differences were observed between the two techniques when the titre of the virus in the plant is high (on spring). Conversely, Real-time is preferable to ELISA for testing tissues with low virus concentration (in the early stages of infection or during summer). Moreover, the accurate and careful observation of symptoms in the field remains crucial in the application of the disease monitoring and eradication programs.
At a glance: Figures
Keywords: plum pox virus, Real time-PCR, DAS-ELISA, stone fruits
Research in Plant Sciences, 2014 2 (2),
Received February 20, 2014; Revised March 20, 2014; Accepted March 23, 2014Copyright: © 2014 Science and Education Publishing. All Rights Reserved.
Cite this article:
- Teshale, Jifara. "Evaluation of Molecular and Serological Diagnostic Techniques for a Large Scale Detection of Plum Pox Virus." Research in Plant Sciences 2.2 (2014): 33-41.
- Teshale, J. (2014). Evaluation of Molecular and Serological Diagnostic Techniques for a Large Scale Detection of Plum Pox Virus. Research in Plant Sciences, 2(2), 33-41.
- Teshale, Jifara. "Evaluation of Molecular and Serological Diagnostic Techniques for a Large Scale Detection of Plum Pox Virus." Research in Plant Sciences 2, no. 2 (2014): 33-41.
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Plum pox virus (PPV) is the agent of sharka, a disease which is considered one of the most devastating of stone fruits in terms of impact and economic importance (Dunez and Sutic, 1988; Nemeth, 1994; Lopez-Moya et al., 2000). Originally described as abnormality of plums in Bulgaria in 1915, PPV was shown to be of viral origin in 1934 (Christoff, 1934). The symptoms of sharka include fruit abnormality and losses of fruits that drop prematurely (Nemeth, 1986).
Susceptible hosts. All Prunus species can be infected by Plum pox virus (PPV), although the presence and intensity of symptoms vary according to species and cultivar, virus strain and sanitary status of the host. The most important susceptible hosts are apricots (P. armeniaca), European and Japanese plums (P. domestica and P. salicina), peaches (P. persica) and their rootstocks (P. persica and P. domestica seedlings, P. cerasifera, P. insititia, P. marianna, etc.). Sweet and sour cherry (P. avium and P. cerasus) can be also affected, although with low incidence and limitedly by the PPV-C strain.
Many other wild and ornamental Prunus species can host PPV: P. spinosa, P. blireana, P. serotina, P. japonica, P. persica f. atropurpurea, P. glandulosa, P. mandshurica, P. mexicana, P. laurocerasus, P. mahaleb, P. davidiana, etc. (James and Thompson, 2006). They may act as symptomatic or, even more dangerously, as symptomless reservoirs of PPV from which the virus can spread to the cultivated crops.
The agent of the disease. The causal agent of Sharka disease is Plum pox virus (PPV), a member of the Potyvirus genus in the Potyviridae family, whose genome has been completely sequenced and characterized (Salvador et al. 2006). PPV virions are long, flexuous and rod-shaped of 660–750 nm in length, formed by a single coat protein (CP) of about 36 kDa arranged helicoidally around one molecule of ssRNA of positive polarity (Brunt et al., 1996).
Based on biological, serological and molecular information available, seven different types or strains of PPV can be recognised: D (Dideron), M (Marcus), EA (El Amar), C (Cherry), W (Winona), T (Turkey) and Rec (Recombinant between D and M), with D and M largely prevailing (Candresse and Cambra, 2006; Serçe et al., 2009).1.1. Detection and Characterization of Plum Pox Virus
Biological methods. Sap inoculation to herbaceous hosts and graft-transmission onto woody indicators represent the most ancient and traditional techniques for PPV and sharka detection. Among the several herbaceous indicators of PPV, Chenopodium foetidum, Nicotiana clevelandi, N. benthamiana and Pisum sativum are the most recommended for diagnosis and characterization studies (Gentit, 2006).
The preferred woody indicator for PPV biological indexing is the peach seedling GF305. P. tomentosa and P. persica cv. Elberta are also recommended as alternative to GF305. Grafting onto woody indicators requires long time for answers if compared with other diagnostic techniques; nevertheless, it remains compulsory to integrate other diagnostic techniques in the application of certification programs of nursery materials.
Serological methods. A decisive impulse to the diagnosis of PPV at a large scale has been given by ELISA. Today, together with polyclonal antisera, many monoclonal antibodies are available as commercial kits. Their use allows to overcome numerous problems associated with polyclonal antibodies, i.e. poor specificity, lack of homogeneity among different batches, strong dependence on the individual immunised animals, low sensitivity. The 5B-IVIA monoclonal antibody of Cambra et al. (1994) has been widely recognized and validated for the universal detection of any PPV isolate. Many strain-specific monoclonal antibodies have been also produced, i.e. 4DG5 for PPV-D (Cambra et al., 1994), AL for PPV-M (Boscia et al., 1997), EA24 for PPV-EA (Myrta et al., 1998) TUV and AC for PPV-C (Nemchinov et al., 1996; Myrta et al., 2000).
Molecular methods. Since their initial description, molecular methods for PPV detection have been increasingly used for their higher sensitivity than ELISA. An eight-fold increase of sensitivity can be obtained with molecular hybridization assay (Varveri et al., 1988; Wetzel et al., 1990), but the use of radioactivity limits the application of such technique for routine analysis. This problem is overcome by the use of polymerase chain reaction (PCR), and in particular by reverse transcriptase-PCR (RT-PCR) which expands the application of the technique by allowing the detection of RNA targets. Although several primers have been described as recognising all PPV isolates, those described by Wetzel et al. (1991) and Levy and Hadidi (1994) are considered as reference for general PPV detection by PCR methods. Sequence data from a number of PPV isolates have permitted the clustering of PPV strains into different groups and the development of group- or strain-specific detection assays (Candresse et al., 1998).
A substantial increase in sensitivity of PPV detection (ca. 250 times) is obtained by the implementation of an immunocapture step prior to RT-PCR (IC-RT-PCR) that simultaneously decreases the concentration of inhibitors (Wetzel et al., 1992).
A significant advance in the detection, quantification and differentiation of PPV strains is by real-time PCR using either SYBR Green I or Taqman chemistry (Schneider et al., 2004; Olmos et al., 2005; Varga and James, 2005). These fluorescent techniques provide a more rapid, sensitive and reliable diagnosis of PPV disease, allow the quantification of PPV viral targets and does not require post-PCR electrophoresis or colorimetric detection.
The use of four easy sample preparation methods (dilution, spot, squash and tissue-print) coupled with highly sensitive real-time RT-PCR has been proposed for routine diagnosis of PPV at a large scale and for the analysis of samples collected during the winter (Capote et al., 2009).
Despite the fact that all of these techniques are quite successful in detecting low titres of the virus, none of these protocols is quantitative in nature.
Because, Biological indexing is time consuming, labour intensive and requires greenhouse space. ELISA depends on availability of PPV polyclonal and monoclonal antibodies. Limitation on the use of monoclonal antibodies may include: (i) unsuccessful detection of PPV during the dormant period of fruit trees; (ii) typing of PPV isolates by subgroup-specific monoclonal antibodies does not always correspond with molecular typing (Candresse et al., 1998). In addition, the evolution of viral serotypes may interfere with reliability of method and only one virus is detected per assay.
That’s why the development of an easy and efficient diagnostic tool to detect this virus and its strains in different period of the year is necessary.
Accordingly, a protocol utilizing a real-time-PCR was found to be simple and reliable for the detection of PPV on large scale.1.2. Objectives
In order to overcome the problem of the low sensitivity and short effective period for PPV detection by biological and serological techniques, the main objective of this study was to verify the possibility to apply the real-time-PCR as an alternative diagnostic method for the PPV detection in large scale survey.
To this aim, Real-time-PCR was compared with the traditional DAS-ELISA technique for detecting PPV during different period of the year and using different plant tissues.
2. Materials and Methods2.1. Sampling and Preparation of Crude Extracts
Samples from apricot (P. armeniaca) and peach (P. persica) adult trees were collected from PPV-infected orchards localized in two main stone fruit growing areas of Apulia and Basilicata regions, in south-east Italy, which had been discovered in the previous year or in the course of the present year.
As infected sources for laboratory analyses (Mediterranean agronomic institute of Bari, Italy), different plant tissues were used which were collected in different period of the year. In particular, dormant cuttings, closed or swelling buds, flowers, leaves and fruits were analyzed in comparative laboratory tests during the complete vegetative season.
All samples were collected from different branches of the tree canopy, labelled, closed in plastic bags and placed in iceboxes at 4°C to be sent to the laboratory for processing. Once in laboratory, the samples were stored in cold room at 4°C before testing by ELISA or Real time PCR (Biorad iQ5 model). All samples (approximately 1 g) were ground into plastic bags containing soft net (Bioreba) using homex-6 machine (Bioreba), in the presence of 1:20 (w: v) phosphate buffer saline (PBS) buffer, pH 7.2, supplemented with 2% (w: v) polyvinyl pyrolidone and 0.2% (w: v) sodium diethyl dithiocarbamate (DIECA). The same crude extract preparations were used for both DAS-ELISA and Real time-PCR.2.2. Tests Carried out during Winter Time on Dormant Plant Tissues
Aim of this test was to verify the possibility to apply, with a certain reliability and efficiency, laboratory tests on dormant plant propagating material, which is the main material used for trade and that favour the long distance spread of the disease. From 5 to 20 budsticks per tree with dormant or swelling buds, from a total of 250 apricot and 137 peach trees were collected during winter time, in two peach and apricot orchards which had been signalled to be infected in the year before and from which, limitedly to the apricot orchard, the single infected trees had been already removed during the summer time of the previous year.2.3. Tests Carried Out during Spring Time on Flowers
Aims of this test were i) to verify the possibility to extend the period for laboratory tests in the field by anticipating the collection of samples from the flowering season; ii) to verify the entity of the eventual natural diffusion of the virus to healthy plants through aphid vectors in the orchards under study. During spring time, from 10 to 15 flowers per tree were collected from the same 250 apricot and 137 peach trees visited in the winter in the two peach and apricot orchards which were found to be infected in the previous year.2.4. Tests Carried out during Spring and Summer Time on Leaves
Aims of this test were to i) compare the sensitivity of ELISA and Real-time tests in detecting the virus in symptomatic and asymptomatic leaves collected from infected trees; ii) identify the parts of the leaves where the virus could be preferentially detected; iii) analyze the variation in the detection capability of the two diagnostic methods in study during the vegetative season. From 20 to 40 fully expanded leaves per tree were collected in four different periods of the vegetative season: twice in Spring (25 March and 03 May) and twice in Summer (11 July and 10 August). Strongly symptomatic, weakly symptomatic and symptomless leaves were collected from each infected tree.
2.4.1. Detection of PPV in Symptomatic and Symptomless Leaves of Apricot
In the spring time, from an infected apricot orchard partially infected (less than 10%), 10 trees showing symptoms only in few sector of the canopy were selected and ideally subdivided in five parts, one symptomatic and four asymptomatic. From these selected trees, 15-20 leaves were separately collected from each of the five parts of the canopy and tested by both DAS-ELISA and real time-PCR techniques.
During summer time (July), the same experiment was repeated in another severely affected apricot orchard where the attenuation or disappearance of the symptoms was mainly due to raising the temperature. Also in this orchard 7 trees showing symptoms only on few sector of the canopy were selected and ideally subdivided in five parts, one symptomatic and four asymptomatic and, similarly to the previous case 15-20 leaves were separately collected from each of the five parts of the canopy and tested by both DAS-ELISA and real time-PCR techniques.
2.4.2. Detection of PPV in Symptomatic and Symptomless Leaves of Peach
In summer period (July), the same experiment was carried out in an infected peach orchard. Also in this orchard 7 trees showing symptoms only on few sector of the canopy were selected and ideally subdivided in six parts, one symptomatic and five asymptomatic and, similarly to the previous case, 15-20 leaves were separately collected from each of six parts to be comparatively tested.
For the same species the experiment was repeated one month later (August) in another peach orchard severely infected. From this orchard a total of 90 asymptomatic leaf samples from six different trees were collected to be comparatively tested in laboratory.
2.4.3. Detection of PPV in Different Parts of the Leaves
In the course of the surveys carried out on July, a total of 20 symptomless leaves per tree from six infected peach and six infected apricot trees were collected. In the case of peach, the leaves were divided into two parts, basal and upper parts, and processed separately in order to know where the virus is more concentrated. The same procedure was applied for apricot; in this case the leaves were divided into three parts: basal (with part of the petiole), medium (lamina) and upper parts.2.5. Tests Carried out during Summer Time on Fruits
Aims of this test were: i) to verify the possibility to extend the period for laboratory tests in the field by delaying the collection of samples to the harvest time; ii) to identify the parts of the fruits where the virus could be preferentially detected. From 5 to 10 fruits per tree, for a total of 40 fruits, 24 symptomatic and 16 asymptomatic, were collected from six infected peach trees during summer time. From each fruit the external epidermis (skin) and the internal pulp were separately tested both by ELISA and Real-time PCR.2.6. Sensitivity Comparative Evaluation Between DAS-ELISA and Real Time-PCR by Analysing Serial Dilutions of Infected Materials
Serial dilutions of virus extracts were prepared in order to assess if the negative results obtained for asymptomatic leaves collected from infected trees was due to the absence or to the low concentration of viruses in the tissues (below the threshold of sensitivity of the diagnostic technique adopted). About 1 g of apricot symptomatic leaves were ground in presence of 2 ml of extraction buffer supplemented with sodium diethyl dithiocarbamate (DIECA). Similarly, 10 g of healthy leaves of apricot were separately ground in 20 ml of the same extraction buffer and distributed in 20 eppendorf tubes (1 ml/eppendorf). One millilitre of symptomatic leaf extracts was added to the first eppendorf tube and well mixed with its content. Then, 1 ml of this mixture was added to the second eppendorf tube, mixed with its content, and 1 ml was taken to be added to the third eppendorf tube. This operation was repeated for all 20 tubes in order to obtain a serial dilution of the virus extract. The same crude extracts were used for both DAS-ELISA and real time-PCR to compare their sensitivity. For real time-PCR the spot procedure was carried out by loading 10 µl into the positively charged nylon membrane, as mentioned before.2.7. Real Time-PCR
The samples were taken from the plastic bags by pipette and poured into sterilised eppendorf tubes. After centrifugation at 5,000 rpm for 3 min, spot procedure was carried out by loading 10 µl of the extract onto positively charged nylon membrane placed in 1.5 ml of sterilised eppendorf tube. To extract the target RNA from the membrane, 100 µl of 0.5% Triton X-100 was added on the membrane, briefly vortexed and stored at -20°C till use (Schneider et al. , 2004; Olmos et al., 2005; Capote et al., 2006).
The reaction cocktail for TaqMan chemistry contained 12.5 µl 2x RT Master mix Taqman (applied biosystem), 0.62 µl RT (applied system), 5.63 µl d H2O, 0.25 µl 100 µM primer P241, 0.12 µl 100µM primer P316D, 0.12 µl 100 µM primer P316M and 0.75 µL 5 µM Taqman probe PPV-DM. After addition of the content of the Taqman chemistries mixture in real time-PCR plate, 5 µl of the target RNA was added in each plate (Capote et al., 2009).
Thermal cycling conditions were as follows: the first two steps were conducted at 39°C for 30 min for reverse transcription, and at 95°C for 5 min for platinum Taq activation. Then 60 cycles of amplification followed at 95°C for 15 sec, and concluded with a cycle at 60°C for 1 min. Fluorescence from FAM reporter was detected at 505-537 nm wavelength. The cycle threshold (Ct) value for each reaction was calculated automatically by the smart cycler (Cepheid) detection software by determining the point (PCR cycler number) at which the reporter fluorescence exceeded 10 times the computer determined standard deviation for background. The size of the PCR product was checked using gel electrophoresis.2.8. DAS-ELISA
For DAS-ELISA (Clark and Adams, 1977) the wells of polystyrene plates were coated with immunoglobulin (usually 1 µg/ml) diluted in coating buffer and incubated at 37°C for 2 hrs. The plates were washed three times for 3 min with washing buffer. Then 100 ml of samples were loaded in each well, and the plates were left to incubate for 2 hrs at 37°C or overnight at 4°C. After washings, 100 µl of enzyme-linked antibodies in conjugate buffer (0.1 µg/ml in PBS with 0.5% bovine serum albumin-BSA) were added in each well and the plates were incubated for 2 hrs at 37°C. After 3 washings, 100 µl of freshly prepared p-nitrophenylphosphate in substrate buffer (1 mg/ml) were placed in each plate.
The plates were incubated at room temperature and photo-metrically measured at 405 nm after 1 and 2 hrs. The ELISA test was considered negative when the absorbance value of the sample was less than two times that of the healthy control and positive if the absorbance of the sample is equal or greater than two times the absorbance of healthy control.
3. Results and Discussion3.1. Tests on Dormant Plant Tissues Carried out in Winter Time
The comparison of ELISA and real time-PCR techniques in PPV detection was carried out in an apricot orchard that had shown a significant infection rate (about 10%) in the previous year. Although all infected trees (about 25%) had been removed during the same year, the presence of other latent infected trees was considered highly probable; therefore, a total of 250 samples consisting in mature bud sticks were collected in winter time from this orchard. Unexpectedly, no infected trees were found after testing all collected samples by both techniques. The complete lack of virus detection from this field could be explained as follows: i) absence of virus inoculums in the field because of the effective eradication of all infected foci in the previous year; ii) low concentration of viruses in the tissues following the very recent inoculation by viruliferous aphids; iii) infected site of the branch escaped the randomized sampling, due to the irregular distribution of the virus in the recently infected trees; iv) the presence of high amount of plant materials like polysaccharides and tannins in buds and phloem tissues interfered with virus detection in laboratory tests.
Similar results were obtained one month later by analyzing the flowers of the same trees in both ELISA and real time PCR tests. Standing this situation, the analysis of this orchard was continued, but it was limited to field surveys for observing symptoms on leaves and fruits. Since all samples were again negative in the tests and did not show any symptom, this orchard was excluded by the planned future experimental trials.
In the same period, 137 samples (bud sticks) were collected from a peach orchard where infected foci had been found in the previous year and no control actions had been taken. In particular, tests interested 50 samples of cv. Big Bang, 36 of cv. Early Top and 47 of cv. Rich May. From these samples the same extracts of buds and phloem tissues were tested by both DAS-ELISA and real time-PCR.
After laboratory analysis, 37.2% of the bud samples resulted positive by ELISA vs 38% by real time-PCR. The same tests carried out using the phloem tissues as infected sources showed slightly lower differences in infection rates (35% and 36.2% in ELISA and real time-PCR, respectively). According to the different varieties tested, the infection rates determined by ELISA were 34% in cv. Big Bang, 2.8% in cv. Early Top and 72.3% in cv. Rich May.
Apart for the case of cv. Early Top, for which the infection rate determined by real time-PCR was significantly higher than that found by DAS-ELISA (13.9% vs 2.8%), no significant differences were observed for the other varieties tested, regardless to the plant tissue examined (buds or phloem). Out of 137 peach bud samples tested, 47 were positive in both DAS-ELISA and real time-PCR techniques. PPV-infection was instead detected only by real time-PCR but not by ELISA in 7 samples and only by ELISA but not by real time-PCR in 4 samples (Table 1).
As mentioned before, the comparison made using 250 flower extracts of apricot as tissues for ELISA and real time-PCR tests proved ineffective, because of the total absence of infection in the selected orchard.
In spring time flower samples were collected for laboratory analysis from the same 137 peach trees tested during Winter. At the time of sampling, the flowers were not fully open and symptoms were not yet visible on them; therefore the collection of flowers (15-20 per tree) was made at random. In ELISA, most of the samples that reacted positive by testing buds and phloem tissues confirmed the same positive results with the flowers. Moreover, very few samples which were negative using buds and phloem tissues became positive using the flowers. In total, 55 samples (40.1%) were positive by using flower tissues, compared to 37.2% of buds.
The same flower extracts used for DAS-ELISA were also used for real time-PCR. In this test PPV was detected in 56 samples (40.9%), only 46 of which were also positive in ELISA (Table 2).
3.3.1. Apricot in Spring
As before mentioned, to be sure that no infected trees were present in the originally selected apricot orchard, in the spring time, after the leaves were well developed, a field survey was carried out for the obervation of symptoms. A second field survey was carried out at harvesting time, to observe the presence of eventual symptoms also on the fruits. Since after accurate and careful surveys no suspected symptom was observed, it was decided to interrupt the planned experimental trials in this orchard.
Therefore, the programmed studies were continued on a new identified PPV-focus in a 3-year-old orchard of cv. Ninfa, in Foggia province, where a certain number of trees showed clear-cut symptoms of sharka disease. In this orchard, together with a few trees heavily affected by the disease, with symptoms uniformly distributed throughout the canopy, the presence of some trees with symptoms limited to few sectors of the canopy was also observed, suggesting that a natural diffusion of the disease was in course likely favoured by aphids.
In spring time, in the above mentioned orchard, ten trees showing symptoms only on few sectors of the canopy were selected and subdivided in five parts, one symptomatic and four asymptomatic. From these selected trees, 15-20 leaves were separately collected from each of the five parts of the canopy and tested by both DAS-ELISA and real time-PCR techniques. In ELISA test, all 10 groups of symptomatic leaves reacted positive; at contrary only one (2.5%) of the other forty asymptomatic groups of leaves coming from the same plants was positive. In real time-PCR the analysis of the same leaf extracts confirmed the 100% detection of PPV in symptomatic leaves, but only in 5 (12.5%) of the other forty asymptomatic groups of leaves coming from the same plants were positive.
3.3.2. Dilution Point
To understand if this result was due to the low concentration (below the threshold of sensitivity of the two diagnostic techniques used in this study) or to the complete absence of viruses in the asymptomatic leaves because of lack of tissue invasion at the early stage of infection, the limit of sensitivity of the two diagnostic techniques was measured by testing a series of dilutions of symptomatic leaves in progressively increased amount of “healthy” leaves. In this test, the ELISA method detected PPV infection up to a dilution point of 1: 28 (1 part of infected tissue in 256 parts of healthy tissue). In the same test, using the same leaf extracts, the real time-PCR technique was able to detect the PPV infection up to a dilution point of 1: 218 (1:364,288), thus confirming the highest sensitivity of this technique compared to ELISA and the ability of this technique to detect the presence of virus infection to a very low concentration.
Based on this result, the hypothesis that in recently infected trees the failure of virus detection in asymptomatic leaves is more likely due to the lack of tissue invasion more than to the low concentration of virus in the tissues seems more plausible. It follows that in similar cases an accurate and careful observation of symptoms in the field becomes more effective than laboratory tests carried out on randomly collected samples. Moreover, very important for the diagnosis of this virus is also how the sample to test is composed, since it must be as much possible representative of the whole tree and thus composed by at least 15-20 leaves collected from the quadrant of the tree canopy.
3.3.3. Apricot in Summer
A similar test was repeated in summer time (July) in an another 4-year-old apricot orchard of the same variety (Ninfa), heavily infected by PPV (more than 70% of infection), but where symptoms were less obvious at the time of the survey because of rising temperatures. Also in this case 7 apricot trees with symptoms visible only on limited sectors of the canopy were selected and ideally separated in five parts, one symptomatic and four asymptomatic.
In ELISA test, all 7 groups of symptomatic leaves reacted positive; at contrary only 6 out of 28 (21.4%) asymptomatic groups of leaves coming from the same plants were positive. In real time-PCR the analysis of the same leaf extracts confirmed the detection of PPV in 100% of symptomatic leaves, and in 18 (64.3%) of the other 28 asymptomatic groups of leaves.
It is evident the difference of this case with the previous one analysed in spring time. In this test the lack of PPV detection in asymptomatic leaves seems due to the low concentration of viruses in leaf tissues because of the effect of the high temperatures. In fact, this effect is more evident with ELISA which is less sensitive, rather than with real time-PCR. It follows that the results of laboratory tests performed in summer on asymptomatic leaf samples, mainly if they are carried out by ELISA, are to be considered unreliable and should be viewed with great caution.
3.3.4. Analysis of Virus Distribution in Apricot Leaf Tissues
An experimental trail was made In summer time (July) to assess the distribution of PPV in the apricot leaf blade. To this aim 20 asymptomatic leaves of six infected trees were collected and singly divided into three parts: lower part including the petiole, central part and apical part (Figure 1). Then all these leaf parts were separately tested by DAS-ELISA. No significant differences in PPV detection by ELISA were observed among the different sectors of the leaf blade. In fact, the virus detection ranged from 11.7% for the central part of the leaves, to 15% and 15.8% for the apical and basal part, respectively
3.3.5. Peach in Spring Time
During the spring time, after the peach leaves were well developed and symptoms of sharka disease became clearly visible, leaf samples were collected from all the same 137 peach trees sampled for the previous laboratory tests. Also for this test the same leaf extracts were used for both real time-PCR (Figure 6) and DAS-ELISA tests. After testing, the responses obtained by the two techniques were practically identical. The general infection level detected was 77.4%, ranging from 47.2% of cv. Early Top, 86% of cv. Big Bang, to 100% of cv. Rich May.
These results showed an infection status of the orchard significantly more impaired than that shown during the previous tests carried out in winter and spring time by using buds and flower tissues (Figure 5) (77.4% vs 37.2% and 40.9%, respectively). It seems unrealistic to assume that this difference is due to the very recent infection. More likely the best diagnostic performance obtained with the leaf material was due to the ease of choosing symptomatic material for sampling (Figure 2). In fact, when asymptomatic leaves were collected from the same infected trees and tested by both DAS-ELISA and real time-PCR tests, the failure of PPV detection was very common. The importance of an accurate and careful visual inspection to choose the best material (symptomatic) for sampling is therefore strongly confirmed also by this test.
3.3.6. Peach in Summer Time
On July, in a 5-year-old peach orchard of cv. Romea partially infected with PPV, seven infected trees showing symptoms on only few sectors of the canopy were selected and ideally subdivided in six parts, one symptomatic and five asymptomatic. From these selected trees, 15-20 leaves were separately collected from each of the six parts of the canopy and tested by both DAS-ELISA and real time-PCR techniques.
By ELISA test, all 7 groups of symptomatic leaves reacted positive; at contrary only 18 (51.4%) of the other 35 asymptomatic groups of leaves coming from the same plants were positive.
In real time-PCR the analysis of the same leaf extracts confirmed the detection of PPV in 100% of symptomatic leaves, and in 28 (80%) of the other 35 asymptomatic groups of leaves, once again demonstrating its major sensitivity compared with ELISA.
On August, 90 leaf samples were collected from a 4-year-old infected peach orchard (100% infected) and tested by both ELISA and real time-PCR. At the time of the survey the symptoms on the leaves were less obvious because of rising temperatures. Out of 90 samples tested 61 (67,8%) were positives by DAS-ELISA and 70 (77,8%) by real time-PCR. In particular, 57 samples were positive in both diagnostic techniques, 4 only by DAS-ELISA and 13 only by real time-PCR.
3.3.7. Analysis of Virus Distribution in Peach Leaf Tissues
An experimental trial was made in summer time (July) to assess the distribution of PPV in the peach leaf blades in order to evidence eventual more concentrated parts to be used in laboratory for testing. To this aim 20 asymptomatic leaves of six infected trees were collected and singly divided into two parts: lower part including the petiole and apical part (Figure 4). Then, all these parts of the leaves were separately tested by DAS-ELISA. After testing, the differences observed in PPV detection from different parts of the leaf blade were not particularly significant, although the basal part of the leaf blade gave a slightly higher number of positive responses if compared with the upper part of the same leaves (41% vs 32%).
On July, 40 peach fruits, 24 symptomatic and 16 asymptomatic were collected from six different infected trees to be tested by DAS–ELISA and real time-PCR (Figure 3). For each fruit the skin and the pulp were separated and singly tested in order to show which part is more suitable for virus detection. By ELISA, out of 40 fruits tested, 28 (70%) were positive when skin was used as tissue, but none with the pulp.
The same extracts were also used for real time-PCR. The test showed that 34 out of 40 samples (85%) were positive from both skin and pulp. All the fruits which were positive by DAS-ELISA were positive by real time-PCR as well, and they included all symptomatic fruits and also some asymptomatic ones (10 samples by real time-PCR vs 4 samples by DAS-ELISA). It is interesting to observe that real time-PCR, contrarily to DAS-ELISA, was able to detect PPV also in the pulp with a good efficiency. Moreover, it is also interesting to confirm the possibility to use the fruits, and in particular their skin, as alternative to leaves in the diagnosis of PPV during summer time.
Plum pox virus (PPV) is the most important and economically devastating pathogen of Prunus species in the world (Lobez-Moya et al., 2000). It can cause losses of fruit yield as high as 95-100% (Nemeth, 1986), therefore, an appropriate monitoring and rapid detection becomes very crucial for its control.
For its easy manipulation, high sensitivity, quick detection and relative low cost, DAS-ELISA is currently used for detecting PPV in large scale surveys in monitoring and eradication programs. Nonetheless, due to the irregular distribution of the virus in infected trees, the fluctuation of its concentration during the vegetative season, and the presence of substances in the plant-tissues of different organs or in different periods of the year that interfere with detection, ELISA has been often questioned for its low sensitivity and reliability, in particular when applied for testing plant materials in non-optimal seasons or in symptomless plants. Unfortunately, other diagnostic techniques which are more sensitive than ELISA, like molecular hybridization or RT-PCR and several its variants, are less suitable for application in large scale surveys due to the laborious and complex manipulation and high costs.
More promising perspectives for solving this problem have been recently provided by the real time-PCR assay, that is highly sensitive and allows to manipulate very easily and quickly many samples without loosing accuracy.
Therefore, this study aimed to compare the performances of real-time PCR and DAS-ELISA in testing PPV-infected apricot and peach trees in different periods of the year and by using different plant tissues. In particular, it was evaluated the potentiality of these two procedures for extending the PPV assays also in those periods of the year that are normally considered unsuitable for testing.
The results obtained in this study have shown that:
Both ELISA and real-time PCR have similar performance in detecting PPV during spring time, when the virus titre is optimal. Therefore, because of its simpler manipulation and low cost, ELISA should be preferred for large scale surveys in this period, even if it is less sensitive than real-time PCR.
The different effectiveness and reliability between the two techniques is more consistent when the titre of the virus decreases, especially in summer time. In this period real-time PCR is without doubts more sensitive and efficient than ELISA, to which it must be preferred for testing in particular symptomless trees. In our experiments, real-time PCR was capable of detecting very low concentration of PPV in plant tissues (up to a dilution point of 1:218 vs. only 1:28 of ELISA).
In summer time, with real-time PCR assay, fruits (both skins and pulp) can be efficiently used as alternative sources to leaves for testing, without a significant decrement in sensitivity. The same organs utilized by ELISA are less performing (only symptomatic fruits react efficiently) and only epidermis should be used.
Tests on bud-sticks and buds are scarcely efficient, probably because of the uneven distribution of the virus in the plant. The same can be said when flowers are used, in particular if they are asymptomatic (as in the case of apricot, plum and several peach varieties) or still closed. In our experiments carried out in an heavily infected peach orchard, about 50% of infected samples were “false negative” when bud-sticks or flowers were used (as it was verified with the subsequent test carried out during spring using leaves). Under these conditions, that are common in the case of very recent infections due to the natural transmission vectored by aphids, the mode of sampling and to compose the samples is of great importance. Samples should be taken from different parts of the tree canopy (in number of at least 10-15) in order to increase the probability to include infected parts in the composed final sample to test.
Not infrequently symptomless materials from infected trees give negative results in laboratory, even when real-time PCR is used. The absence of symptoms in infected trees can mainly be due to: i) the low concentration of virus particles in the tissues due to very early stage of infection or to the effect of high temperature; ii) the complete absence of viruses in complete sectors of the canopy in case of very recent transmission by aphids. In this last case the absence or the rare detection of PPV is very common, as we verified in a test on apricot trees carried out during spring time (2.5% and 12.5% with ELISA and real-time, respectively). When the absence of symptoms was due to the effect of the high temperature, the successful detection from peach leaves in two tests carried out in summer was 51.4% and 67.8% by ELISA, whereas it was 80% and 78.9% by real-time PCR. The same test with apricot leaves gave 21.4% of successful detection by ELISA and 64.3% by real-time PCR. It can be concluded that the laboratory tests carried out on symptomless materials are only partially reliable for detecting infected plants, since very high is the percentage of infected plants which remains undetected (about 80% for apricot and 30-50% for peach by using ELISA, and about 35% for apricot and 20% for peach by using real-time PCR). In any case the use of basal parts of the leaves is also suggested since it slightly increases the capacity of detection of the virus by ELISA. For all these cases, the accurate and careful observation of symptoms on each single tree still remains critical for an efficient detection of PPV in monitoring and eradication programs.
In general, real time-PCR has given proof to be a valid alternative to ELISA for large scale detection of PPV during different period of the year, and especially during Winter and Summer, when the virus concentration in plant tissues tends to decrease. Its application to the analysis of large numbers of samples can contribute to control the sanitary status of commercial Prunus trees and to prevent the spread of sharka disease.
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