Plum pox virus has been transmitted by at least 20 aphid species, although only a few are considered important vectors. The vectors that are considered important vectors in the northeastern United States include: the black bean aphid (Aphid fabae), the spirea aphid (Aphid spiraecol), the black peach aphid (Brachycaudus persicae), and the green peach aphid (Myzus persicae).

Method	Instructions	NAPIS Survey Method
Visual	Collect symptomatic plant tissue. The hierarchical sampling method is recommended PPV (Hughes et al., 2002).	00550 - National Plum Pox Virus Survey

No specific signs are present.

Symptoms of PPV can be conspicuous or very subtle on stone fruit trees. Symptoms vary in type and severity with the strain of the virus, host, cultivar, environmental factors, and the timing of infection. Diagnostic symptoms occur mainly on leaves and fruits in the United States. In general, leaf symptoms include vein yellowing or light green to yellow rings. Foliar symptoms may develop during the cooler temperatures of spring and fall but fade during the hot summer months. Symptoms of PPV occur sporadically and often are not apparent until three or more years after infection. Newly infected trees are rarely symptomatic.

Plums: Pale green or light yellow chlorotic spots, blotches, bands, rings, or line patterns may occur on the leaves. They are difficult to see in the bright sunlight. Leaf symptoms are most easily seen on the fully expanded leaves from late May/early June. These symptoms are often irregularly distributed and may appear on only a few branches or leaves. Plum fruit symptoms depend on the original color of the fruit. Dark-skinned fruits show bluish, necrotic rings, which may be sunken. Pale-skinned fruit show uneven ripening, blotching, and rings. Necrotic tissue may extend through the flesh to the stone, on which a reddish necrotic ring may develop. Plum fruits are often deformed. Also, some plum cultivars can drop fruit prematurely.

Peach: The leaf symptoms of PPV on peach are distinctive. Affected leaves are distorted when they first unfold, having a wavy edge and a slight twist, and the veins show pale green or bright yellow flecks or lines. These symptoms disappear as the leaves mature. Peach fruit may develop lightly pigmented rings or line patterns that result from the convergence of several rings. Peach fruit, however, may have paler colored rings and lines than those found in plums. Peaches are generally more susceptible to damage from the disease than plums. Flowers on PPV-infected peach trees may exhibit color breaking but only on cultivars with large showy flowers. Color-breaking appears as darker pink stripes on the flower petals.

Apricot: Show lighter symptoms than plum or peach. Apricot fruits may be misshapen, turn brown or become necrotic and may have rings on the surface of the seed.

Almonds: Show few leaf symptoms. Infection is often symptomless.

Cherry: Pale green patterns and rings appear on the leaves. Fruits are slightly deformed with chlorotic and necrotic rings, notched marks, and premature fruit drop.
Note: PPV strain D, which occurs in the United States, is not known to naturally cause infection in cherry.

The visual symptoms accompanying the reduction in sugar content make the affected fruit unmarketable.

The approved screening protocol for the field is the PPV Enzyme-Linked ImmunoSorbent Assay (ELISA). The work instruction is available upon request. The work instruction describes detection of PPV using the ELISA kit from Agdia Inc., which detects six known PPQ strains/subgroups: PPV-C, PPV-D, PPV-EA, PPV-M, PPV-Rec, and PPV-W in leaves, fruit, and flowers.

PPV symptoms are sometimes difficult to distinguish from other diseases and may be confused with rusty spot (Podosphaera spp.) of peaches and nectarines and bacterial canker as well as insect-related problems such as damage from thrips, white apple leafhopper, and San Jose scale. Nutritional deficiencies and pesticide damage can also be confused with PPV symptoms.

Indicator Hosts: Prunus persica cv. GF305, P. tomentosa, Chenopodium foetidum, Pisum sativum, and others can be inoculated and monitored for diagnostic symptom development.

Antibodies: Monoclonal antibody 5B-IVIA (Cambra et al., 1994) allows for the universal detection of PPV. Strain-specific monoclonal antibodies have also been developed for both the D and M strains/serotypes (Cambra et al., 1994; Boscia et al., 1997). These enzymes, however, are not suited for differentiating other serotypes (e.g., El-Amar and Cherry) (Candresse et al., 1998). Polyclonal and monoclonal antibodies have also been produced for the PPV-C and PPV-EA strain (Boscia et al., 1997; Crescenzi et al., 1997; Myrta et al., 1998; Myrta et al., 2000).

ELISA: Clark and Adams (1977) developed the first ELISA test and included Plum pox virus as an application of this technology to plant viruses. Strain-specific antibodies are available (see section above).

Lateral Flow Device: Mumford et. al. (2010) describe a lateral flow device for on site detection of PPV. The on site kit, which contains a one-step lateral flow device and a simple, bottle extraction system, can give a result in three minutes.

Immunochromatographic Assay: Byzova et al. (2010) raised two monoclonal antibodies that recognized strains PPV-D, M, and C. The authors developed a 10-minute immunochromatographic assay for PPV with a detection limit of 3 ng/ml. The assay demonstrated good compatibility with the data obtained via ELISA.

Molecular: Sequence analysis of PCR fragments corresponding to the C-terminal part of the PPV coat protein gene has allowed identification of a molecular polymorphism correlated to serotype of the PPV isolates (Candresse et al., 1994; Candresse et al., 1995). Initial results have indicated that an RsaIrestriction fragment length polymorphism (RFLP) located in this region could be used for PCR amplification, to discriminate between D and M serotypes of PPV (Bousalem et al., 1994; Wetzel et al., 1991). More recently, a cluster of non-coding, third-base mutations on five consecutive codons located around the RsaI RFLP site was found to show excellent correlation with the viral serotype (Candresse et al., 1995). This observation was used as a basis for direct PCR typing of isolates belonging to the D and M serotypes of PPV (Candresse et al., 1994).

Levy and Hadidi (1994) utilized a simple and rapid procedure for processing PPV infected plant tissue (Gene Releaser) for use with a specific 3' non-coding region RT-PCR assay. The 3' non-coding region was used, because Asian Prunus latent potyvirus, a newly identified latent potyvirus in Prunus spp., may react positively with PPV-coat protein primers, in Southern blot hybridization with a PPV coat protein clone, and in ELISA with PPV polycolonal antiserum (Hadidi and Levy, 1994).

Immunocapture PCR (IC-PCR), reverse transcriptase PCR (RT-PCR), RT-PCR with RFLP, PCR-ELISA, print-capture PCR, and integrated RT-PCR/nested PCR have been used to detect PPV and to type strains/serotypes of PPV (Wetzel et al., 1991; Wetzel et al., 1992; Olmos et al., 1996; Olmos et al., 1997; Olmos et al. 1999; Poggi Pollini et al., 1997; Hammond et al., 1998; Nemchinov et al., 1998; Staniulis et al., 1998; Szemes et al., 2001; Glasa et al., 2005; Papayiannis et al., 2007; Olmos et al., 2008). Szemes et al. (2001) developed a RT-PCR/nested PCR technique for the simultaneous detection of PPV-D, M, EA, and C.

Faggioli et al. (1998) compared three different techniques to prepare PPV viral RNA for RT-PCR: 1) an immunocapture technique using a specific antiserum, 2) a silica-capture method using a non-specific matrix, and a simple and rapid RNA extraction. All three techniques allowed for the successful amplification and detection of PPV, but the silica capture method was less effective.

Candresse et al. (1998) compared an indirect double antibody sandwich ELISA using monoclonal antibodies for PPV-D and PPV-M with specific PCR assays or RFLP analysis of PCR fragments. Overall, the authors found an excellent correlation between the results of the ELISA and PCR assays for PPV-D and PPV-M. IC-PCR has been shown to be about a thousand times more sensitive than ELISA (Candresse et al., 1995).

Sanchez-Navarro et al. (2005) developed a multiplex RT-PCR for the detection of eight stone fruit viruses, including PPV. Jarosova and Kundu (2010) used a single-tube multiplex RT-PCR to detect PPV, Prune dwarf virus, and Prunus necrotic ringspot virus. Both methods included an internal control.

Real-Time PCR: Schneider et al. (2004) developed a real-time, fluorescent, RT-PCR reaction assay for the detection of PPV in the Smart Cycler (Cepheid). Varga and James (2005) developed real-time multiplex assay utilizing SYBR Green technology to detect and differentiate PPV-D and PPV-M types in woody and herbaceous plants. Olmos et al. (2005) used Taqman technology in real-time assay for the universal detection and quantification of PPV in plant material and aphid vectors. The sensitivity of the real-time RT-PCR assay was 100 times higher than nested RT-PCR and 1000 times higher than ELISA and conventional RT-PCR.

In the United States, the disease was recorded in Pennsylvania in 1999 followed by New York and Michigan in 2006. The disease has since been eradicated in Michigan and Pennsylvania. Eradication efforts are continuing in New York (Levy et al., 2000).

Seven strains of PPV (D, M, El-Amar, C, W, T, and Rec) have been identified worldwide based on their biological, serological and molecular properties to date. PPV-M and PPV-D are the most widespread. All occurrences in the United States have been identified as strain D (PPV-D).