Fruit trees are grown worldwide, mainly as a food source, and Prunus
spp. are one of the most popular cultivated trees. The genus Prunus
includes almonds, apricots, cherries, peaches, plums, and nectarines. In the United States, the main producers of Prunus
spp. are the states of California, Washington, Oregon, South Carolina, Georgia, Michigan, and New Jersey (https://www.nass.usda.gov/index.php
Although economic losses due to viral infection in Prunus
spp. are difficult to quantify, viruses can cause losses by reducing plant vigor and growth, delaying fruit ripening, and causing graft and compatibility issues. Viruses can also remain latent, later, causing plants to grow slowly, produce smaller fruit, and have a reduced lifespan, but often these detrimental impacts may go unnoticed unless crops are visibly damaged [1
]. Reduction in yield and poor product quality from some viruses can be severe and lead to tree removal. Some major viruses of Prunus
spp. include apple chlorotic leaf spot virus (ACLSV), cherry green ring mottle virus (CGRMV), cherry leaf roll virus (CLRV), little cherry virus-1 and -2 (LChV-1 and -2), prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV). Cemballi et al. [2
] estimated that the United States sweet cherry and clingstone peach industries could save $
11,191,460 and $
5,580,877, respectively, adopting a virus protection program.
The genetic diversity of plant viruses is well known (reviewed in [3
]). For example, divergent variants of LChV-1 and LChV-2 have been characterized via high throughput sequencing (HTS), which affects the epidemiology and symptomatology associated with these viruses [5
]. PNRSV and PDV isolates can be classified in several phylogroups based on their coat protein (CP) or RNA-dependent RNA polymerase (RdRp) genes [7
]. This genetic diversity makes it difficult to design and maintain sensitive assays that will reliably detect different virus isolates. Nucleic acid specific detection of viruses is a very useful technique to determine if a particular virus is present in a plant. Methods relying on PCR require sequence specific primers which may not amplify a virus sequence if the virus has nucleotide differences at the primer binding site. Given the genetic diversity of viruses, it is possible to miss detection of virus strains if the PCR assay is not specific for a variant present in nature.
Efficient and reliable laboratory diagnostic tests are critical in determining viral infection in Prunus
spp. While several diagnostic methods are available for viral detection (e.g., biological indexing and ELISA), the advantages of using real-time reverse transcription PCR (RT-PCR) to detect viruses have been documented. The development of real-time RT-PCR assays led to superior sensitivity, speed, reproducibility, and limited risk of contamination compared to end-point RT-PCR [9
]. The main feature of real-time RT-PCR is that DNA amplification is detected in real time as RT-PCR is in progress by the use of a fluorescent reporter, thus, the reporter signal strength is directly proportional to the number of amplified copies [11
]. These characteristics often make it the method of choice in routine diagnostics. Virus testing of imported propagation materials into the United States has been the most important measure used to prevent the introduction and spread of viruses [2
], and real-time RT-PCR is one of the diagnostic tools employed by inspection agencies. There are two types of real-time RT-PCR systems. The first is based on a generic non-sequence-specific double-stranded DNA-binding dye such as SYBR Green, and the second is based on sequence-specific DNA hydrolysis probes [11
]. In this study, we used TaqMan hydrolysis probes with a FAM dye label on the 5′ end and a minor groove binder (MGB) and nonfluorescent quencher on the 3′ end.
Here, currently available real-time RT-PCR assays for different Prunus
-infecting viruses (Table 1
) were evaluated, and in many cases updated or redesigned to accommodate additional sequence diversity that was not available at the time the assay was originally designed. In the case of viruses with no published real-time RT-PCR assay, a new assay was designed. Thus, 15 new or updated real-time RT-PCR assays were developed during this study. In most cases, these assays utilized multiple primers and probes for detecting all known virus variants. Comprehensive evaluation and compact design (i.e., use the minimum number of primers to cover the genetic diversity) of so many assays were made possible because of purpose built Python scripts. Subsequently, all assays were empirically validated using previously known infected plant material. Lastly, for additional validation, a Prunus
germplasm collection, representing different accessions originating from 53 countries, was screened with the real-time RT-PCR assays.
This study exhaustively evaluated the genetic diversity represented by currently available Prunus
fruit tree virus assays. We developed new and updated real-time RT-PCR assays to improve representation of current genetic diversity. These assays were designed with the dual goals of being compact, and at the same time, incorporating a complete picture of the known genetic diversity for high efficiency and sensitivity. New assays were designed to the most conserved region present in each virus species, which may involve the CP, RdRp, triple gene block 1, or the 3′ untranslated region. Among virus isolates, pairwise sequence similarity within assay regions varied between 88.5% to 100% (Table 4
). In order to generate all these assays, custom scripts were utilized to accelerate and simplify assay design (e.g., sequence alignment and primer/probe design were completed in a single process). This also allowed us to use multiple variant matched primers and probes instead of single degenerate pairs. The smaller and more uniform primers in these assays are an attempt to ameliorate the lower efficiency degeneracy can lead to [12
]; additionally, the use of degenerate primers may result in a major number of primers per reaction in comparison with our new assays.
Prunus samples from two collections, FPS and CPCNW, were used as virus sources to evaluate the updated or new assays. HTS analyses indicated that most of these samples (i.e., 69 out of 87) were infected with at least one of the 15 targeted viruses, revealing mixed infections in several samples (i.e., 20 samples). As a result of this evaluation, an agreement between real-time RT-PCR assay and HTS was obtained. To further validate the real-time RT-PCR assays, we collected and tested samples from the NCGR, which includes Prunus spp. accessions from a wide range of geographical regions. Although the actual virus diversity in the NCGR samples was not characterized by HTS, we detected 12 of the 15 viruses in 54.6% of the trees, suggesting that the PCR-based assays are robust. Thus, all the updated or new assays were tested against multiple isolates of each virus, except for CRMaV, which was identified in only one instance by both real-time RT-PCR and HTS.
During the initial validation of the real-time RT-PCR assays using samples previously analyzed by HTS, Ct values ranged from 12 to 28 and similar Ct values were obtained during the survey in the NCGR (Table S2
). For CGRMV, CLRV, and PNRSV assays, there were a few cases where Ct values were >30. These samples were re-analyzed (i.e., extraction and testing were repeated) and confirmed to be negative. We hypothesize that these high Ct values were due to cross-contamination from strongly positive samples that were present in the initial processing. Consequently, any amplification after 30 cycles should be further investigated and verified.
In the United States, growers have adopted different methods for the control of viral diseases in fruit trees, including (i) the adoption of virus-tested propagation material and (ii) the eradication of infected trees [2
]; all the viruses here investigated are part of the clean plant certification program. In that sense, new advances in real-time RT-PCR have significantly improved the detection of pathogens, allowing quick, sensitive, and precise identification compared to other historically used detection methods (e.g., end-point PCR, ELISA, and biological indexing). Moreover, real-time RT-PCR can be used to determine the number of virus copies present in a sample (i.e., virus quantification). In addition, it has the potential to be multiplexed with other assays, increasing testing efficiencies by identifying different viruses during the same reaction or by including an internal control. Thus, the development of highly sensitive real-time RT-PCR assays with broad-range detection capacity is needed for large scale testing of Prunus
species that may be infected by the genetically diverse viruses included in this study. The assays developed here can help the clean stock programs and the fruit tree industry by facilitating early detection of virus-infected material. Likewise, Fotiou et al. [15
] just published a new real-time RT-PCR for plum pox virus, which is considered as one of the most important pathogens in fruit trees and currently quarantined in the United States.