Investigation of Plum pox virus in Different Tissues of Apricot and Plum Trees

MATERIALS AND METHODS

Bark, flower parts (sepal, petal, stamen, gynoecium and pollen), fruit, seed and leaf samples (as a control) were collected from PPV M and D infected apricot and plum trees (5 of each-unknown cultivars) in Ankara between 2002 and 2004. Fifty samples per infected tree were collected which were then tested using indirect ELISA.

The indirect ELISA tests were completed using duplicate wells in polystyrene microtitre plates using the methods outlined by Uyemoto et al. (1989). Samples were extracted using a coating (carbonate) buffer (pH 9.6) containing 0.045 g L^-1 sodium diethyl dithiocarbamate (Na-DIECA), 20 g L^-1 polyvinyl pyrrolidone-25 (PVP) and 2 g L^-1 bovine serum albumin (BSA), 1/20 w/v. Plates were incubated with plant extracts for 2 h at 37°C. The polyclonal PPV antiserum was diluted in a phosphate-buffered saline (PBS) that contained 20 g L^-1 polyvinyl pyrrolidone-25 (PVP) and 2 g L^-1 bovine serum albumin (BSA), 1/1000 v/v. The plates with the diluted antisera were loaded within 3 h and kept at 37°C. The alkaline phosphatase-conjugated goat anti-rabbit antiserum was later diluted in the latter buffer (1/50000 v/v) and the plates with conjugate were incubated for 3 h 37°C. The ELISA values were obtained by measuring the absorbance of the substrate at 405 nm, after incubation with an alkaline ρ-nitrophenyl phosphate in substrate buffer (0.5 mg mL^-1) at room temperature.

The investigation of seed-borne transmission of PPV involved the collection of apricot and plum seed taken from severely infected trees (25 seeds from each tree). The seeds were then stratified in perlite at 4°C (Stokes, 1965). After germination the seeds were transferred to the pots. Seedlings from stratified seeds were periodically tested for PPV infection using indirect ELISA over a 2-year period.

RESULTS AND DISCUSSION

Bark, flower parts (sepal, petal, stamen, gynoecium and pollen), fruit, seed and leaf samples were collected from PPV M and D infected apricot and plum trees and the following results were obtained. As well, the seedlings were evaluated for PPV infection.

Bark: Samples were taken in February, from the internodes and included the phloem and all surrounding tissue. PPV was detected in the samples of both the apricot and plum trees (Table 1). Adams (1978), Dosba et al. (1986), Adams et al. (1998) and Polak (1989) found similar results in studies that employed similar ELISA techniques. Adams (1978) reliably detected PPV using plum bark samples taken in January and February. According to the data obtained by Adams et al. (1998), PPV was detected in bark samples collected in December, January and March. Polak (1989) reported that PPV was reliably detected in bark of apricot trees. Thus, there is no contradiction between earlier investigations and the results of this study.

Flower: PPV was found in sepals, petals and stamens (Table 1). Petals are frequently used as a source of virus inoculum from woody plants but the study by Clark and Adams (1977) indicated that petals from Prunus insititia (damson plum) were a poor source of PPV compared with sepals and gynoeciums. A similar distribution of the virus was found in the flowers of Prunus domestica (plum) and Prunus ceracifera (myrobalan plum) (Adams, 1978). In contrast, Rankovic and Vuksanovic (1981) did not detect PPV in the petals of PPV-infected apricot trees.

In this study, PPV was also detected in stamens but was not detected in the pollen and gynoecia of the trees investigated (Table 1), a result supported by both Adams (1978) and Dosba et al. (1986). Trifonov (1965) first discovered the presence of the virus in the pollen of infected trees. Macovei (1970) pollinated healthy plum trees with the pollen of diseased plum cv. Agen then characteristic virus symptoms around the pollinated flowers the following year. Coman and Cociu (1976) found 20-80% pollen transmission according to the susceptibility of the plum cultivars. The above mentioned reports indicate that, there are documented cases of positive transmission of PPV by pollen. However it is generally thought that PPV is not transmits by pollen.

Fruit: PPV was detected in the fruit of infected apricot and plum trees (Table 1). Llacer et al. (1986) found that plum fruit was the most reliable parts of the plant for detecting PPV by ELISA. This is in agreement with the results obtained by van Oosten (1970) who used plum fruits for the mechanical transmission of PPV to herbaceous plants. Adams (1978) found that PPV could be detected on the fruit of plum trees. Similarly, Dosba et al. (1986) also detected PPV from on the fruit, bark, buds, flowers, stamens and leaves of apricot trees. Present results support these findings.

Table 1:	Absorbance values of different tissues of the PPV infected stone fruit trees
H: Healthy, I: Infected, Gyno.: Gynoecium

Pit: Pit rings were observed in many of the apricot fruits, whereas no symptoms were observed on plum pits. PPV was detected in apricot pits, but it was not detected on plum pits (Table 1). We found no literature regarding detection of PPV in pits.

Seed: In this study, PPV was not detected in the seeds (Table 1) and seedlings. Seed transmission in Prunus is not known to occur with the D strain, but there are some reports of seed transmission with the M strain. Seed transmission on apricots was first reported by Szirmai (1961). Coman and Cociu (1976) found 8-10% seed transmission depending on the susceptibility of the plum cultivars. Nemeth and Kölber (1982) found PPV in 23.2-79.4% of seeds originating from diseased apricot trees and reported 3.4-13.9% PPV infection in the 2- year-old seedling progeny of diseased trees. Eynard et al. (1991), Triolo et al. (1993) and Pasquini et al. (1998) detected PPV in apricot seeds; Glasa et al. (1999) found PPV in plum seeds and Pasquini et al. (2000) found PPV in apricot and peach seeds. However, seedlings from infected seeds have never showed symptoms and have been found to be PPV-negative by serological and molecular assays (Schimanski et al., 1988; Eynard et al., 1991; Triolo et al., 1993; Dulic-Markovic and Rankovic, 1997; Myrta et al., 1998; Pasquini et al., 1998; Glasa et al., 1999; Pasquini et al., 2000). Most researchers think that the virus is inactive during seed germination. The most commonly held view of scientists working on PPV in many countries is, however, that seed and pollen transmission does not occur and the differences in results obtained are probably a result of the differentiated character of the various PPV strains (Myrta et al., 1998; Desvignes, 1999; Pasquini et al., 2000).

The apricot and plum seedlings were monitored for symptom expression over two years and tested using ELISA. Fortunately, none of the test specimens were ELISA positive showing no PPV symptoms. The virus could be detected on apricot pits (not plum pits) not the seeds indicating that PPV is not seed-transmitted.

In conclusion, bark tissue was found to be an opportune tissue in the detection of the PPV during the winter in Ankara. Despite the lack of antigen in some tissues, whole flowers are acceptable sources of antigen for ELISA. Some of the stone fruit varieties have no or less distinct symptoms on the fruit (Elibuyuk and Erdiller 1995) and the absorbance values of fruits increased as the symptom became more severe (Adams, 1978). Therefore the fruit of these trees is not a good source of PPV and thus not suitable for the detection of the virus. Further, the non-existence of PPV in pollen, seeds and seedlings is a reassuring result in terms of dissemination of the virus in Ankara as our results indicate that PPV was not transmitted via pollen or seeds in Ankara. We did find that the disease was transmitted by mealy plum aphids (Hyalopterus pruni) (Elibuyuk, 2003).

Our results indicate that PPV was detected consistently in extracts of most types of tissue tested using ELISA. However, because the distribution of the virus is uneven in infected trees (Nemeth, 1986), small numbers of samples from each tree may be inadequate to detect PPV. Therefore, a sufficiently large number of samples must be taken if ELISA assays are to be employed in the detection of this disease.

Our results were found to be similar to the found by other investigators. Nevertheless, the presence and concentration of the virus in the infected plants vary considerably with plant cultivar, the age of the plant and climatic conditions (Nemeth, 1986, Desvignes, 1999). Furthermore, because the distribution of PPV in infected trees is uneven, erratic results might be obtained in detection. Even though ELISA is a convenient detection method, molecular techniques (e.g., RT-PCR) has improved PPV diagnosis, as these techniques are more sensitive than serological tests and can detect very low virus concentrations in plant tissues (Wetzel et al., 1992). However, even if PPV is found in pollen and seeds, seed and pollen-transmission studies should still be performed.