Recent advances in characterization and diagnosis of stone fruit tree viruses

Abstract

Around seventy-five viruses have been reported to infect stone fruits which heavily reduce its yield and fruit quality, resulting in major economic losses. Viruses are very contagious and have devastating consequences on the host plants. In most of the stone fruit cultivars the viral infection is mostly symptomless and has the capability to infect a wide range of hosts. The most critical part of controlling these stone fruit viruses is proper diagnosis. Traditional approaches are regarded insufficient for identifying plant viruses due to less precision, and accuracy. To close the existing gap, researchers created specific, sensitive, and effective molecular techniques as a diagnostic tool for plant virus detection, which have the potential to improve disease management decision-making by overcoming several limitations of traditional methods. Due to the high risk of continuous spread of these viruses and for routine diagnosis to avoid further infection, management strategies must be incorporated. With the development of molecular techniques for viral disease identification and characterization, it became attainable to gain an enhanced and detailed understanding of their prevalence and genomic organization. This is an updated review on the viruses of stone fruits, their modes of transmission, genome organization, detection, and diagnosis.

Introduction

Stone fruits belongs to family Rosaceae are fleshy fruits with thin epicarp (outer skin), a layer of edible flesh underneath the skin called mesocarp, a hard ovary wall and a highly lignified endocarp known as pit or stone which encloses the seed (Kole and Abbott 2012; Wani et al. 2024). The major commercial stone fruits include apricot, sour cherry, peach, nectarine, European plum, sweet cherry, and Japanese plum. These stone fruits have a diverse geographic origin extending from eastern Europe to China (Ogawa 1991). The demand for stone fruits is emerging since they have high nutraceutical properties packed with vitamins, minerals, carbohydrates, carotenoids, lipids, phenolic, organic acids, and other metabolites which are essential in our daily diet (Meena et al. 2021). Stone fruits hold a pivotal position in contributing to the agricultural economies of multiple countries. The major stone fruit growing countries are America, China, Australia, Iran, Afghanistan, Central Asian countries, France, Portugal, Italy, Greece, New Zealand, and India (Malik et al. 2020). Stone fruits are more vulnerable to freezing damage in the early spring due to their early blooming nature as compared to pome fruits. After planting, production for stone fruit trees begin between the third and fourth year followed by its commercial output lasting for roughly 15 to 25 years. Aboveground, stone fruit trees consist of a fruit-yielding scion cultivar and the underground rootstock develops into the root system (Lockwood and Coston 2005; Rasool et al.’ (2020). They reproduce asexually through root cutting, tissue culture, grafting or budding on different rootstocks (Hamid et al. 2025; Hartmann and Kester 1975; Mandal et al. 2025; Marina et al. 2020; Nabi et al. 2024). These all techniques have helped in the international and national spread of numerous plant diseases especially viral diseases. Throughout history, stone fruits are prone to various fungal, bacterial and viral diseases and most of these are common as they are closely related, and this makes them one of the main production constraints (Desvignes et al. 1999, Sweet 1979; Wani et al. 2024). Among the various diseases of stone fruits, viruses are considered to be a major threat to stone fruit production globally (Barba et al. 2015; Umer et al. 2019). In addition to reducing productivity, viral infections can impair the form, size, and quality of fruits, resulting in severe economic losses (Hadidi and Barba 2011; Jones and Naidu 2019). Some of the economically significant viruses infecting stone fruits include apple mosaic virus (ApMV), apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), cherry leaf roll virus (CLRV), plum pox virus (PPV), prune dwarf virus (PDV), peach mosaic virus (PMV), prunus necrotic leaf spot virus (PNRSV), etc. (Aparicio et al. 1998, Noorani and Khan 2020). Viruses infecting stone fruits are widely disseminated, causing large losses in both quality and quantity of fruits. They are often subtle, frequently overlooked, and untreated (Wani et al. 2024). These viruses sometimes may not produce any visible symptoms (latent) in their hosts and frequently result in delayed development of plants, produce fewer fruits, fruits attain smaller size, reduced life span, change the product's composition and/or additional frequently overlooked effects (Hadidi and Barba 2011; Singhal et al. 2021). These viruses can produce a variety of symptoms, such as twisting or deformation, necrotic and chlorotic regions, mottling, rolling, shot holes and unusual colour patterns of leaves to general decline in vigour and productivity (Fig. 1) (Wani et al. 2025b). Fruits may exhibit shape distortions, modifications such as mottling, ringspots, and line patterns, as well as decreased size and quality (Hadidi and Barba 2011). Furthermore, compared to annual crops, viral infections in perennial crops are frequently more severe and incur no cure (Chandel et al. 2013; Manzoor et al. 2025; Wani et al. 2024). Therefore, creating efficient and long-lasting management methods to stop the spread of viral infections depends on early identification and correct diagnosis (Hamid et al 2025, Ibaba and Gubba 2020). An analysis of the existing literature was conducted on the characterization and diagnosis of viruses affecting stone fruit trees. To initiate this review, we gathered pertinent articles from the Web of Science by using keywords such as “stone fruit viruses, Potyviridae, Bromoviridae, Comoviridae, Flexiviridae, Betaflexiviridae, Closteroviridae, Gene Silencing, High Throughput Sequencing, Metagenomics, RNA Interference, and Viral Metagenomics”. This resulted in a collection of 2000 relevant articles published between 2010 and 2025, which provided comprehensive bibliometric data that served as input for VOSviewer analysis. Out of 6,051 keywords, 438 met the threshold, with a minimum occurrence of 5. For each of these 438 keywords, we calculated the total strength of co-occurrence links with other keywords. Those keywords with the highest total link strength were then selected for further analysis. This analysis evaluates the frequency of keyword usage and citation metrics within the selected articles. Furthermore, we created a network visualization map that illustrates international research in this area, highlighting the global distribution and collaboration within the field (Fig. 2).

Fig. 1.

A Distortion. B–C Chlorotic and mosaic patterns. D Necrotic spots and shot holes. E unusual colour patterns, F Twisting and rolling of leaves

Fig. 2.

A Network visualization of co-occurrence of keywords, illustrating the relationships and frequency of usage among key terms providing insights into the concentration and distribution of keywords within the analyzed literature B the global distribution and collaboration in stone fruit tree virus research. Nodes represent keywords, with larger nodes indicating greater total link strength, while connections show co-occurrence relationships. The visualization highlights key research areas and the interconnected efforts of the global research community

Role of plant genome encoded proteins in viral infection

Plant genome encoded proteins play a vital role in facilitating the infection process of stone fruit viruses (Ilardi and Nicola-Negri 2011). These viruses are dependent on the host proteins at almost all the stages of their life cycle- its entry, replication, movement and immune evasion. Viruses like plum pox virus (PPV) use the host’s eukaryotic initiation factors proteins (eIF4E) for translation inside the host cells (Wang et al., 2002). The host cytoskeleton related proteins and membrane trafficking components help the viruses to move between plant cells which causes systemic infection and symptom development (Koziel et al. 2017). These viruses also manipulate the plant stress related proteins like heat shock proteins (HSP70) to help in stabilizing viral replication complexes (Hýsková et al. 2021). Viruses also target the proteins involved in the RNA silencing machinery like Argonaute (AGO) and Dicer like proteins (DCL) to disrupt the gene silencing pathway and create an environment favorable for infection (Singh et al. 2021). These mechanisms need to be further understood to develop advanced management technologies.

Major viruses infecting stone fruits

It has been discovered that around 75 viruses from various species and families infect stone fruit plants in which identification of nearly 40 novel viruses have been facilitated by High Throughput Sequencing (HTS) technology in the last decade (Umer et al. 2019). The nature of the costs incurred by these viruses differs greatly from those attributed to other diseases, which means that they are accountable for larger economic losses than is typically acknowledged. Losses go unrecognized and addressed because they are typically more subtle and less obvious. Some viruses cause negative consequences such as slowed plant development, fewer fruit production, changed product composition, shorter lifespans, or other undetectable effects even when they are mainly latent in part of their hosts (Barba et al. 2015). On stone fruit hosts, mixed infections with prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and apple mosaic virus (ApMV) are frequent (Çağlayan et al. 2011; Costa et al. 2022b). The cherry green ring mottle virus (CGRMV) is latent in the majority of prunus populations (Barba et al. 2015). ACLSV has been connected to severe fruit deformities, production losses, graft incompatibility, and bud necrosis in some stone fruit cultivars. A disease called “pseudopox” disease is caused by a few strains of ACLSV in apricots and plums. This disease is characterized by protuberances and depressions that distort the fruit (Barba et al. 2015). Cherry mottle leaf virus (CMLV) is among the most damaging disease affecting cherry cultivars in certain areas (Nemeth 1986). The highly destructive tomato ringspot virus (ToRSV) of the Nepovirus genus is responsible for decline, and stem pitting decline in Prunus spp. Another intricate and serious viral infection that significantly affects the fruit yield of infected cherry trees worldwide is the little cherry disease. Little cherry virus 1 and 2 (LChV-1 and LChV-2) are associated to this disease. More research is needed to determine how most of these new viruses affect stone fruits. Multiple viruses belonging to different families infect stone fruits (Table 1), some of the major ones are discussed. In addition to the mentioned viruses above, few viroids have also been reported in stone fruits so far like Peach Latent Mosaic Viroid (PLMVd) Apple scar skin viroid (ASSVd) and hop stunt viroid (hsvd) (Al. Rwahnih et al. 2001; Costa et al. 2022a; Fiore et al. 2016; Kaponi et al. 2012, 2010; Maghraby et al. 2007; Stanbekova et al. 2025).

Table 1.

Stone fruit viruses detected using NGS technology

Family	Genus	Species	Host plant	Technology type	Platform	Speed	References
Betaflexiviridae	Capillovirus	Apple stem grooving virus	Apple, Pear	HTS of RNA	Illumina NextSeq 500	High	Costa et al. 2022a, b
		Cherry virus A	Cherry, Apricot	Metagenomes of RNA	Illumina HiScan 2000	High	Baráth et al. 2018, Costa et al. 2022
	Foveavirus	Apple stem pitting virus	Apple, Quince, Pear	HTS of RNA	Illumina NextSeq 500	High	Costa et al. 2022
		Asian prunus virus 1, 2 and 3	Peach			High
		Apple green crinkle associated virus	Apple, Pear			High
	Tepovirus	Prunus virus T	Pear			High
	Trichovirus	Apple chlorotic leaf spot virus	Apple, Quince, Peach, Plum			High
		Peach chlorotic leaf spot virus	Peach			High
		Apricot pseudo- chlorotic leaf spot virus	Peach	HTS of RNA	Illumina HiSeq 2000	High	Jo et al. 2018
	Robigovirus	Cherry green ring mottle virus	Cherry	HTS of RNA	Illumina NextSeq 500	High	Costa et al. 2022
		Cherry necrotic rusty mottle virus	Cherry, Peach	HTS of RNA	Illumina NextSeq 500	High	Costa et al. 2022, Jo et al. 2018
Bromoviridae	Ilarvirus	Apple mosaic virus	Apple	HTS of RNA	Illumina NextSeq 500	High	Costa et al. 2022
		Prune dwarf virus	Cherry, Plum		Illumina NextSeq 500	High	Chirkov et al. 2023, Costa et al. 2022)
		Prunus necrotic ringspot virus	Cherry, Plum	HTS of RNA	Illumina NextSeq 500	High	Costa et al. 2022 Chirkov et al. 2023, Jo et al. 2020
Closteroviridae	Ampelovirus	Little cherry virus 2 and 1	Cherry, Apricot	Metagenomes of RNA	Illumina HiScan 2000	High	Baráth et al. 2018, Costa et al. 2022)
		Plum bark necrosis stem pitting-associated virus	Cherry, Peach, Plum	HTS of RNA	Illumina NextSeq 500	High	Costa et al. 2022, Jo et al. 2020
	Velarivirus	Little cherry virus 1	Cherry, Plum	HTS of RNA	Illumina NextSeq 500	High	Costa et al. 2022, Jo et al. 2020)
Comovirinae	Nepovirus	Cherry leaf roll virus	Cherry		Illumina NextSeq 500	High	Costa et al. 2022
Luteoviridae	Luteovirus	Apple luteovirus 1	Apple		Illumina NextSeq 500	High	Costa et al. 2022
		Nectarine stem pitting associated virus	Cherry			High
		Peach associated luteovirus	Peach			High
Partitiviridae	Alphapartitivirus	Pear alphapartitivirus	Pear			High
Phenuiviridae	Rubodvirus	Apple rubbery wood virus 1 and 2	Pear, Apple			High
Tymoviridae	Marafivirus	Nectarine virus M	Cherry			High
		Peach virus D and T	Peach		Illumina HiSeq 2000	High	Jo 2018

Potyviridae

One of the most important virus causing huge yield losses in stone fruit production is known as plum pox virus (PPV) which comes from the genus Potyvirus and family Potyviridae with an entire genome size of roughly 10 kb (García et al. 2014; James et al. 2013). Plum pox or Sharka disease is one of the widely distributed viral disease of Prunus species described first-time during 1st World War in Bulgaria in 1915 (Atanassov 1932). It is caused by plum pox virus (PPV) and significantly lowers fruit production as well as fruit quality (Cambra et al. 2006; Llácer and Cambra 2006). Because of its economic impact, it is a well-studied plant virus. However, some of its protein functions have been predicted by analogy with other Potyvirus members (Scholthof et al. 2011).

PPV was first identified in plums but other stone fruits, including peaches, apricots, sweet and tart cherries, were subsequently discovered as other hosts of PPV (Llácer and Cambra 2006). Despite stringent quarantine regulations, it had spread almost across the globe and has been detected in all the continents. Due to its existence in India, trade restrictions were imposed on a local, national, and international scale (Rimbaud et al. 2015; Šubr and Glasa 2013). PPV is made up of non-enveloped, flexuous, filamentous positive-sense RNA virus particles that are about 750 nm in length and 15 nm in width. PPV is polyadenylated at 3΄end and contains virus-coded protein at 5΄ end (Laín et al. 1988; Riechmann et al. 1989) (Fig. 3a). It codes for single large polyprotein of about 355.5 kDa (Riechmann et al. 1992; Simón-Buela et al. 1997) and three viral protease domains having autocatalytic proteolysis property leading to the formation of particular mature polypeptides and proteins thus presumed to be multifunctional.

Fig. 3.

ORFs of different stone fruit viruses. a ORF of plum pox virus (PPV); b ORF of prunus necrotic ringspot virus (PNRSV); c ORF of prune dwarf virus (PDV) d ORF of cherry leaf roll virus (CLRV); e ORF of tomato ringspot virus (ToRSV); f ORF of apple chlorotic leafspot virus (ACLSV); g ORF of apple stem grooving virus (ASGV); h ORF of apple stem pitting virus (ASPV)

In addition, PPV has a variety of strains and at least ten monophyletic strains (D,C, EA, M, T, W, An, Rec, CR, and CV) have been reported. The severity of the disease and its accompanying symptoms differ across various strains (Chirkov et al. 2018; García et al. 2013; Maejima et al. 2020). Prunus plants infected with PPV may display a variety of symptoms. However, incubation period (the time before symptoms appear) and the degree of severity is determined by the viral strain, host species, nutritional state of the host plant, age of the cultivar, infection period, environmental factors (such as temperature) and the infection period (Llácer and Cambra 2006). In most of the cases, visible symptoms often do not appear until 3–4 years after infection (Travis et al. 2000). Most susceptible cherry, peach, apricot, and plum cultivars display foliar symptoms in the form rings or spots which are green, light or yellowish in color, or mottling on the leaves (Maejima et al. 2020). Infected plum fruits frequently have abnormal lines, bands and poxes on their surface in addition to being deformed. Apricot fruits feature colored bands on their epidermis, and the stones have light rings or dots. The symptoms on peach fruits are confined to its skin, with formation of diffuse bands and pale rings before the fruit ripens (Barba et al. 2011). The appearance of chlorotic bands or “halos” on the fruit epidermis is possibly the most distinctive sign of PPV infection. Deformation of fruits may occur with irregularity in its shape and the development of necrotic or brown dead areas which causes fruit to drop early and possibly, cause tree mortality as well (Llácer and Cambra 2006). On the leaves and fruits of infected trees, visual symptoms may or may not appear, although decrease in crop production occurs even on trees that show no symptoms. In addition, PPV reduces the productive life of infected trees due to the decrease in fruit quality and grade, eventually damaging the entire host tree. The lower portions of the branches are frequently where the virus is found, but not at the tips. Once symptoms start to show up on branches, they continue to show up in the following years (Travis 2001). The green peach aphid (Myzus persicae), which colonizes most stone fruits, is one of the most effective vectors of PPV, spreading the virus over short to medium distances in a non-persistent manner (Gildow et al. 2004; Pasquini and Barba 2006; Travis 2001). Movement of infected planting material or plant components by human activity is the primary cause of long-distance PPV dissemination. Mostly, grafting and other vegetative replication techniques are used to spread the virus. (Damsteegt et al. 2007). Grafting is one the most popular means of Prunus propagation, therefore infected rootstocks, budwood and scion wood can be transported over great distances (Damsteegt et al. 2007; Hunter et al. 1958; Mandal et al. 2025).

Bromoviridae

A major chunk of viruses infecting stone fruits belong to this family, one of the most important one is prunus necrotic ringspot virus (PNRSV). According to (Cochran and Hutchins 1941), PNRSV was initially identified as the “peach ring spot virus” in peach plants that displayed significant ring spots. Globally present, the PNRSV is a dangerous virus that affects a wide variety of woody plants. It causes mosaic disease in apples, roses, and plums, as well as ring spot diseases in peaches, cherries, roses, and hops (Mink 1993). Several additional names for the disease are hop B virus, red currant necrotic ringspot virus, sour cherry necrotic ringspot virus, rose vein bending virus, and rose yellow vein mosaic virus. The PNRSV has been first reported from peach orchards of USA and is categorised under the genus Ilarvirus (Cochran and Hutchins 1941). When it comes to fruit trees, ilarviruses are typically regarded as latent diseases since the virus particles are comparatively unstable in tissue extracts, which has led to an underestimation of their economic significance (Pallas et al. 2012). PNRSV has a non-enveloped, isometric, and bacilliform structure with three genomic strands (tripartite) and is made up of a single-stranded, positive sense RNA genome. The particles are 23, 25, and 27 nm in size and contain RNA1 of 3.3 kb, RNA2 of 2.5 kb, and RNA3 of 1.9 kb, respectively (Zhang and Qu 2015) (Fig. 3b) Each of these genomic regions has a highly conserved 3′ end and a less conserved 5′ end. All 3 genomic RNAs function as messenger RNAs: RNA1 and RNA2 encode proteins that are necessary for the replication of viruses, while RNA3 encodes the coat protein and potential mobility (Fauquet et al. 2005). Since PNRSV is a latent virus, it can infect any host and remain symptomless, but it can also cause a wide range of distinctive symptoms (Hammond 2011) (La Torre-Almaraz et al. 2014). The severity of the disease is influenced by the type of virus isolate, the host cultivar, and the synergistic interactions of PNRSV with other viruses like prune dwarf virus (PDV). In most woody hosts, PNRSV infection first results in shock, which is followed by the development of persistent symptoms. Following the shock period, the trees usually seem fairly normal, but in reality, the virus keeps replicating and grows in concentration every year (Uyemoto and Scott 1992). On freshly grown leaves, PNRSV first manifests as chlorotic patches and rings that subsequently turn necrotic. Eventually, the necrotic tissues separate from the leaf, giving the leaf a ragged or "shot hole" appearance. Additional symptoms of the virus include mottling, vein banding or yellowing, yellow mottle, linear and mosaic patterns, and chlorotic bright yellow discolorations in the form of line and oak leaf patterns, which are occasionally accompanied by pronounced deformation of the leaf blades (Brunt et al. 1996) (Kapoor and Handa 2017). A significant amount of decline in trees and stunting is also seen, and a substantial proportion of the tree canopy has creamy spots developing on it. Symptom diversity leads to major diseases in nurseries and complicates identification and diagnosis in the field (Bonilla et al. 2023; Scott et al. 2001) examined the impact of PNRSV virus on peach growth, yield, and quality and discovered that it reduces tree growth by 12 to 70 percent and results in fruits with lower soluble sugar content by 5 to 70 percent. PNRSV has been shown to cause notable yield losses in sweet cherries and peaches, with reported losses of up to 100 percent in each case, depending on the host. Reduced bud-take in nurseries, a 10–30 percent drop in fruit growth, and a 20–60 percent reduction in fruit output due to delayed fruit maturity are further effects of PNRSV (Pallas et al. 2012). PNRSV is easily transmitted using standard plant propagation techniques (Cole et al. 1982) and root grafting in orchards and hence, is widely dispersed throughout the world. PNRSV is easily spread by seeds, and it is also present in pollen grains (Mink 1993). Additionally, it can spread by mechanically inoculating herbaceous hosts with infected sap (Hammond 2011) or by several thrips species transporting contaminated pollen to healthy plants (Amari et al. 2009; Digiaro et al. 1992).

Prune dwarf virus (PDV) is another virus, belonging to the same family, that affects stone fruit trees globally and is commercially significant, mostly affecting almond, peach, plum, and sweet and sour cherry trees (Uyemoto and Scott 1992). Thomas and Hildebrand discovered prune dwarf virus in plums from New York State for the first time in 1936 . It is a member of the class of viruses with a tripartite single-stranded positive sense RNA genome belonging to the genus Ilarvirus (Codoñer et al. 2005). The replicase, also referred to as the P1 protein, is encoded by the RNA1. The proper functioning of the replication complex and the anchoring of viral RNA at the assembly site are significantly influenced by this protein. While RNA3 is bicistronic and codes for motility and capsid proteins (Szyndel and Paduch-Cichal 1997), RNA2 codes for the replicase subunits of RNA-dependent RNA polymerase (RdRp), which is in charge of replicating viral RNA (Codoñer et al. 2005; Codoner et al. 2006). Whereas the coat protein is produced by a subgenomic RNA4 molecule (sgRNA4) and is also involved in genome activation, movement protein is directly created by RNA3 (Koziel et al. 2015; Pallas et al. 2012) (Fig. 3c). PDV shows great variation in symptoms depending on the virus strain, climate, host species, and cultivar (King 2012). Small, thin, or curled leaves with short internodes, chlorotic ring patches, and even gum leakage from some hosts, such as apricots, are typical symptoms of PDV-infected plants (Koziel et al. 2015). Peach cultivars are said to experience mild stunting from PDV, with their leaves turning a darker green color and being more upright than those of uninfected plants (Çağlayan, Ulubas-Serce, Gazel and Varveri 2011). The PDV-infected peach trees exhibit significant internode stunting, which causes emerging shoots to form rosettes, which in turn reduces plant development and fruit production (Scott et al. 2001). PDV is readily dispersed by pollen and is manually transmitted among plants. Greber et al. demonstrated that thrips (Frankliniella occidentalis) may also transmit PDV, but only in lab conditions where the transmission level was close to 20 percent (Greber et al. 1992, Koziel et al. 2015). Using certified nursery stock and removing any diseased trees that have shown symptoms during survey and monitoring are simple ways to manage PDV. Viral-free rootstocks and scion wood are to be utilized in nurseries, and viral signs should be closely observed. Viral transmission can be substantially inhibited by clean cultivation (Greber et al. 1992)(Kozieł et al. 2017).

Bromoviridae also has another important virus infecting stone fruits, called apple mosaic virus (ApMV), which was initially identified in apples (Bradford and Joley 1933). As per (Murphy et al. 1995), the virus belongs to the genus Ilarvirus. Its multi-component tripartite positive sense ssRNA genome contains three RNAs, RNA1, RNA2, and RNA3, which have respective sizes of 3.476, 2.979, and 2.056 kb. These RNAs are contained in icosahedral virions, with a a diameter of 26–35 nm (Lakshmi et al. 2011). RNA 3 is bicistronic and codes for both coat and movement proteins, while RNA 1 and 2 are monocistronic and encode nonstructural proteins involved in viral replication (Lakshmi et al. 2011). Stone fruit hosts are among the several woody species that are infected by ApMV. ApMV infections have been reported sporadically and frequently in conjunction with PNRSV and PDV infections in stone fruit hosts (Paunovic et al. 2011). In susceptible cultivars, the virus results in decreased growth and yield losses; however, exact data regarding the virus's economic impact is not yet available. The characteristic yellow line pattern, vein clearing, and oak leaf pattern are seen on the leaves of infected stone fruits. Typically manifesting around the onset of summer, symptoms have occasionally been limited to a few evenly spaced leaves on the plants (Paunovic, Pasquini and Barba 2011). In case of almond, its infection results in failure of blossom and leaf bud growth therefore also known as almond leaf failure (Desvignes et al. 1999). ApMV is serologically similar to PNRSV and serological studies mistakenly classify it as PNRSV (Petrzik and Lenz 2011). It spreads only by vegetative propagation of rootstocks, planting material especially bud- and graft- wood from infected mother trees and slow natural spread via roots in nurseries has been reported. The virus has neither any known insect vector and nor it is pollen transmitted (Digiaro, Savino and Terlizzi 1992).

Comoviridae

Among the many plant viruses under this family, cherry leaf roll virus is an important one which was initially identified in Persian walnuts (Juglans regia) in North America by (Schuster and Miller 1933), and in sweet cherries in England by Posnette in 1955 (Cropley 1961). Afterwards, reports of CLRV infecting a large number of deciduous trees, shrubs, ornamentals, herbaceous plants, and weeds have been made worldwide. Furthermore, CLRV has been documented in a variety of hosts from over 36 families (Woo 2014). It belongs to the genus Nepovirus and the family Comoviridae. The CLRV is single-stranded, positive-sense isometric bipartite virus with two RNA molecules, RNA-1 and RNA-2 (Fig. 3d). Its RNA genome is 15 kb in length (Murant et al. 1981), and both RNAs are independently encapsulated in isometric particles (Jones 1973), measuring 28 nm in diameter and have angular outlines (Schuster and Miller 1933). The CLRV is also known as elm mosaic virus, ash mosaic virus, walnut blackline disease, sambuccus ring spot and yellow net virus. Depending on the host species, CLRV strain, and the climatic conditions these symptoms can change. CLRV-infected cherry trees exhibit symptoms such as delayed flowering, leaf rolling, bunching of shoots, yellowing and chlorosis of leaves, and 91–98 percent crop losses (Büttner et al. 2011). Since freshly infected trees with CLRV may not exhibit noticeable symptoms for several years, providing a continuous source of inoculum that aids further spread, challenging to stop the virus from spreading. In a number of host species, CLRV is easily spread by grafting, seed, and pollen (Jones 1973). Seed transmission of CLRV is especially important because it gives the virus a natural route of dispersal in the environment (Stace-Smith and Hamilton 1988). Unlike other nepoviruses, CLRV is not thought to be transmitted by nematodes, despite the lack of information regarding any insect vectors. Nematodes such as Xiphinema and Longidorus species have been reported to transmit CLRV (Jones et al. 1981); however, replication and verification of these findings have not been possible (Cooper and Edward 1980; Wang et al. 2002).

Another major virus infecting stone fruits grouped under this family is tomato ring spot virus (ToRSV). In 1936, Price initially reported the tomato ringspot virus, from Nicotiana species.It is one of the damaging viral infections of stone fruits that results in large yield losses. It is classified under genus Nepovirus (Sanfaçon and Fuchs 2011). ToRSV is a bipartite genomic virus that is encapsulated in isometric particles having a diameter of 25–28 nm (Fig. 3e) (Bellardi and Marani 1984; Zaitlin and Palukaitis 2000). Yellow bud mosaic or prunus stem pitting may appear, depending on the virus strain and scion wood combination. As the virus advances into scion wood and eventually impairs tree growth, yellow bud mosaic causes foliar symptoms that gradually extend throughout the canopy. As spurs, twigs, and small branches are killed by the virus, an appearance of naked limbs increases from the base of branches upward (Moini 2010). This disease is characterized by spongy bark that is excessively thick near the tree's base (Moini 2010). The virus exhibits transmission through mechanical inoculation, yet grafting and dagger nematodes (Xiphinema spp.) are notably more efficient vectors for its dissemination (Forer et al. 1984).

Flexiviridae

One major virus affecting stone fruit production is apple chlorotic leaf spot virus (ACLSV) which is known to infect most rosaceous fruit trees and belong to the family, Flexiviridae. According to (Mink and Shay 1959), the virus is widely dispersed and early discovery was from the USA following transmission to Malus platycarpa. Apple, pear, quince, peach, plum, apricot, and cherry are among the fruit tree species that are known to be infected by this virus (Watpade et al. 2012) (Martelli et al. 1994) identified as ACLSV belonging to the genus Trichovirus. The genome of ACLSV is a single-stranded positive sense polyadenylated RNA molecule measuring 7555 kb, although it lacks a poly-A tail. ACLSV are flexuous, often filamentous particles measuring 720 × 12 nm (Brunt, Crabtree, Dallwitz, Gibbs and Watson 1996). Three open reading frames make up ACLSV, with ORFs 1, 2, and 3 encoding proteins with molecular weights of 216.5, 50.4, and 21.4 kDa, respectively (German et al. 1990) (Fig. 3f). ACLSV exhibits a significant degree of molecular diversity, and several viral isolates with varying degrees of pathogenicity have been reported. The primary reasons of ACLSV being a significant virus economically are its global distribution and its potential to seriously impair ability to undergo grafting in some Prunus species, resulting in severe problems in nurseries. Despite causing latent infection in many hosts, ACLSV can cause variable severity symptoms in different susceptible cultivars, many rootstock/scion combinations or in mixed infection with other latent viruses (Watpade et al. 2012). ACLSV infection also leads to different syndromes in different hosts, such as russet ring of apple, ring pattern mosaic of leaves in quince and pear, Malus platicarpa dwarf and leaf deformation of several ornamental species of apple. Instead of being symptomless, ACLSV can split bark on some stone fruits. It can also cause severe fruit deformations, yield reduction, bud necrosis, false plum pox, also known as pseudopox, peach dark green sunken mottle, and severe leaf deformation (Myrta et al. 2011). This pseudopox disease is called such because it resembles the real plum pox disease and is distinguished by protuberances and depressions that distort the fruit. It is sometimes mistaken for the “sharka” disease caused by plum pox virus. It has been reported in apricot and plum as well (Sutic et al. 1999). Although the ACLSV's natural route of transmission is still unknown, grafting has been shown to be the primary means of field dissemination (Brunt et al. 1996). As of right now, no natural vector is known to spread ACLSV (Yoshikawa 2001), and there is no evidence that the virus is spread via pollen or seeds. The virus is latent in the majority of infected trees, which raises the possibility of accidental spread and distribution of planting material contaminated with ACLSV. Thus, for ACLSV to be properly managed, it is necessary to understand its host range (Ghanem et al. 2002). Coinfection with apple stem pitting virus and/or apple stem grooving virus is frequently associated with ACLSV infection. Therefore, only trees grown from clean, virus-tested buds and rootstock liners should be planted in order to ensure a long-lasting, healthy orchard that produces good harvests with high-quality fruit. To stop ACLSV from spreading, propagation material should only come from clean, virus-tested trees (Ghanem, Stino and Samia 2002).

Betaflexiviridae

Cherry virus A (CVA) is one of the viruses that affects stone fruits globally and is commercially significant (Jelkmann 1994; Komorowska and Cieślińska 2004). This virus is a member of the genus Capillovirus and family Betaflexiviridae In 1941, Cochran and Hutchins reported the disease's initial cases on sweet cherries in Germany that were afflicted with small cherry disease. There have been reports on peaches, apricots, plums, and both sweet and sour cherries from different regions of the world (Simon-Loriere and Holmes 2011, Wani et al. 2024). Cherry virus A (CVA) has positive-sense RNA genome made up of two open reading frames (ORFs). The movement protein is encoded by ORF2, but the RNA-dependent RNA polymerase, helicase, methyltransferase, and coat protein are all encoded by a protein that is encoded by ORF1(Martelli et al. 2007). While the consequences of CVA on fruit output and quality are mostly still unknown, it is believed that infected plants do not get diseased. However, in apricot leaves, CVA was observed to show vein-clearing symptoms (Koinuma et al. 2016). Severe symptoms or graft incompatibility in susceptible rootstock and scion combinations can arise from the mixed infection of CVA with other viruses, such as the prune dwarf virus or little cherry virus (James and Jelkmann 1997a, b) (Marais et al. 2008). CVA is graft transmissible, latent, and has a global distribution, while there are no vector known to transmit it (Svanella-Dumas et al. 2005).

Another economically significant virus is the Apple stem grooving virus (ASGV) which also belongs to the same family, was first discovered in 1965 by Lister et al. in American Malus sylvestris (Nemeth 1986). The virus is known to infect a range of significant crops, including stone fruits, and it has a broad host range (Clover et al. 2003; Hirata et al. 2003; Tang et al. 2010; Yoshikawa et al. 1993). Virus particles are flexible filaments with a clear cross banding, measuring 12 nm in width and helical symmetry. The genome is a 6,496 nucleotide unipartite positive sense single-stranded RNA made up of two overlapping open reading frames, ORF1 and ORF2, which together encode a 241-kDa polyprotein and a 36-kDa protein, respectively (Yoshikawa et al. 1993) (Fig. 3g). When an infected is grafted on a sensitive rootstock, the symptoms can be variable and lead to severe stem pitting and grooving of the xylem, brown line, and graft union abnormalities. In nurseries, symptoms are less vigorous than the healthy ones (Massart et al. 2011; Yoshikawa 2001). Infection rate can be as high as 80–100 percent and yield losses may reach 40 percent(Hirata et al. 2003). There is no known insect vector for ASGV and is transmitted by infected propagative material including budding and grafting (Nabi et al. 2018).

The 1940s saw the initial reports of another fatal virus causing apple stem-pitting disease in American apple cultivars. ASPV is another member of the family Betaflexiviridae and genus Foveavirus. ASPV is a single-stranded positive RNA (+ ssRNA) virus with a genome of about 9300 nucleotides that codes for the 5′ and 3′ untranslated regions and five open reading frames (ORFs). The viral RNA-dependent RNA polymerase (RdRP) is encoded by ORF1, triple gene block proteins are encoded by ORF2-ORF4, and the viral coat protein is encoded by ORF5 (Fig. 3h) (Ma et al. 2019). The virus particles have helical symmetry and are flexuous and filamentous, about 800 nm in length and 12–15 nm in width. Many plant species, especially stone fruits, are infected by ASPV. Most cultivars show no symptoms at all, but depending on the plant species, cultivar, and viral isolate, it can cause a range of symptoms in some susceptible cultivars of fruit trees and indicator plants, including xylem pits, epinasty, vein yellowing decline, leaf red mottling, pear necrotic spot, or fruit stony pits (Jelkmann 1994; Mathioudakis et al. 2006, 2010; Wu et al. 2010). Although there isn't a known natural vector for ASPV, it can spread through grafting and/or contaminated propagative material (Jelkmann and Paunovic 2011).

Betaflexiviridae accommodates most of the major stone fruit viruses with another major one being apricot latent virus (ApLV). First reports of ApLV was in Moldavia in 1993, associated with the Bulgarian-introduced “Silistra N4” cultivar of apricot. (Martelli et al. 2007). Its genome is monopartite positive-sense ssRNA with a 3'poly (A) tail (Grimová and Ryšánek 2012) and most of its molecular characteristics are closely related with ASPV (Grimová et al. 2010; Youssef et al. 2011). In spring chlorotic lesions followed by green spots appeared on the leaves, but it remains mostly latent in the natural host (apricot) from which it was reported for the first time (Grimová and Ryšánek 2012). The pathogen, P. armeniaca, is known to only naturally infect apricot trees. However, it has been experimentally spread to multiple Prunus species through grafting. ApLV has been identified to date from several countries, including Moldavia, Italy, Turkey, Iran, France, Palestine, Egypt, Czech Republic and Spain, despite its low prevalence (Grimová et al. 2010). But due to mild or asymptomatic infection, ApLV is still regarded as a pathogen of minor importance. Although, there is no insect vector known for ApLV, it mainly spreads through grafting and thrives in the host materials used for propagation (Grimová and Ryšánek 2012).

Two other viruses under this family are cherry mottle leaf virus (CMLV) and cherry green ring mottle virus (CGRMV). CMLV first identified in Oregon, USA, in 1920, impacts several economically important fruit cultivars including apricot, peach, and cherry (McLarty and Pathologists 1935). This virus features a single-stranded positive-sense RNA genome measuring 8.018 kilobases, encapsulated within filamentous virions spanning dimensions of 640–800 × 12 nm. The virus causes leaves to become deformed and mottled with chlorosis. Fruits from diseased trees are small, flavourless and show delayed ripening, which lowers their market value. For certain cherry cultivars, CMLV is the most serious disease (James and Jelkmann). It spreads by bud/scale mite Eriophyes inaequalis, grafting, and budding (Nemeth 1986). Cherry green ring mottle virus (CGRMV) was reported for the first time from Michigan USA in 1937 on sour cherry plants (Prunus cerasus) and later in 1951 it was identified as a viral disease, although it is normally latent in most of the Prunus species like peach, apricot, nectarine (Barba et al. 2015), but in case of cherry, latent infections of CGRMV are more common. Cherry CGRMV, which belongs to the genus Robigovirus, has a positive sense ssRNA genome of 8.372 kb and is encapsulated in filamentous virions that are between 1000 and 2000 nm in size. The diseased leaves have green patches and circles set against a background of mottled yellow, and the plants frequently produce unmarketable fruits (Jelkmann and Paunovic 2011). CGRMV spreads through vegetative propagation methods like budding or grafting, employing infected planting material. Additionally, natural root grafting between healthy and diseased trees facilitates its transmission. Notably, CGRMV does not disseminate via seed transmission and lacks any identified insect vector (Fridlund 1967).

Closteroviridae

Little cherry disease (LChD) is a multifaceted and potentially life-threatening viral ailment associated with little cherry viruses 1 and 2 (Jelkmann and Paunovic 2011). These two distinct viruses, identified as LChV-1 and LChV-2, belong to the genera Velarivirus and Ampelovirus, correspondingly, within the Closteroviridae family. They possess single-stranded positive-sense RNA genomes, with sizes of approximately 16.930 and 15.050 kilobases, respectively. These genomes are enclosed within elongated virions ranging from 1786 to 1820 nm in length. There are just a few species of Prunus that these viruses may infect, and cherry is said to be the most common host worldwide. The fruit production of the affected trees is greatly impacted by these viruses. Each virus causes different symptoms on the fruits such as a drastic reduction in colour, size and taste as well as a complete loss of crops with stunted vegetative growth. Some cultivars have either mild fruit deformation or some may have resistance to it. These viruses can cause single or combined infections; however, infections combining LChV-1 and LChV-2 can result in severe symptoms (Hadidi et al. 2011). Both viruses linked to LChD are easily transmitted by grafts. The apple mealy bug (Phenacoccus aceris) and grape mealy bug (Pseudococcus maritimus) are the most important vectors of this disease and also carrier of LChV-2 (Mekuria et al. 2013). However, LChV-1 is not considered to be carried by any vector (Jelkmann and Paunovic 2011).

Detection methods for viral diseases of stone fruits

Historically, the detection of plant viruses relied on visual inspection, which is the most straightforward and simplest method available (Fig. 4). While viral infections in plants manifest in diverse ways, several common symptoms are frequently observed (Mehetre et al. 2021). Initially, staining techniques were employed which made it possible to visualise visible areas of infection (Senshu et al. 2011).

Fig. 4.

Conventional and recent detection methods for viral diseases of stone fruits. (PCR: Polymerase Chain Reaction; RT-PCR: Reverse Transcriptase Polymerase Chain Reaction; ELISA: Enzyme Linked Immuno-sorbent Assay; DBIA: Dot Blot Immunobinding Assay; TBIA: Tissue blot Immunobinding Assay; LAMP: Loop-Mediated Isothermal Amplification) (Created with BioRender.com)

Other visual detection methodologies that emerged with time includes visible light imaging, chlorophyll fluorescence imaging, hyperspectral imaging, and thermal imaging techniques (Mutka and Bart 2015). However, these methods are not reliable as they are highly driven by environmental and biological factors besides lacking uniformity in their results. While valuable, accurately interpreting these tests requires a high level of expertise. Viral symptoms can be deceptively similar to those caused by mineral deficiencies, and misdiagnosis is a potential pitfall. Furthermore, these techniques are not applicable in asymptomatic cases, where infected plants display no visible symptoms of infection. Electron microscopy was the first ever technique to be used for visualization of viruses, specifically tobacco mosaic virus (TMV) in 1939. Among the two types of electron microscopy, transmission electron microscopy (TEM) is mainly used for viruses as it possesses higher resolution. Negative staining followed by visualization in TEM can be performed simply for quick identification with precision. PNRSV, PPV and PDV are some of the viruses that have been detected using TEM method (Jakab-Ilyefalvi et al. 2011). An advanced adaptation of electron microscopy is cryo-electron microscopy (cryo-EM) which elucidates 3D structures of bio-macromolecules at sub-nanometre resolution. This imaging technique gained high recognition after being honoured with the Nobel Prize (Kühlbrandt 2014). The three-dimensional configuration of macromolecular assemblies can be best explored using cryo-electron microscopy (Cryo-EM). This development in electron microscopy has now made a breakthrough to visualize mechanisms involving viral particle assembly, genome organisation etc. (Richert-Pöggeler et al. 2019). The 3D model of plum pox virus has successfully been visualised using cryo-EM (Jevremović et al. 2023).

After which, serological methods were developed and used for the diagnosis of viruses because of their many advantages in its reaction specificity allowing virus quantification even in the presence of impurities or other host derived materials that might be present in the sample. A frequently utilized serological assay for effective stone fruit virus detection is the enzyme-linked immunosorbent assay (ELISA) (Iralu et al. 2025; Wani et al. 2025a). This method has demonstrated efficacy in detecting numerous stone fruit viruses, including plum pox virus (Adams et al. 1997), apple chlorotic leaf spot trichovirus (ACLSV) (SerÇE and Ertunç 2005), apple mosaic virus (ApMV), prunus necrotic ringspot virus (PNRSV) (Candresse et al. 1997), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV) (Çağlayan, Ulubas-Serce, Gazel and Varveri 2011), peach rosette mosaic virus (Stobbs and Van Schagen 1996), among others.. Dot blot immune-binding assay (DBIA) is another immunological assay which can detect virus even at minimal sample volume. Stone fruit viruses like PNRSV (Abdel-Salam and Mokbel 2021) and PDV (Narayanasamy and Narayanasamy 2011) have been detected using DBIA. Tissue Blotting Immunoassay (TBIA) is another practical, cheap and rapid serological adaptation used to detect stone fruit viruses like ACLSV (Rana et al. 2011) and PDV (Abou-Jawdah et al. 2004). However, all there are multiple advancements made in the realm of detection and management with the aim to improve the sensitivity, specificity and speed.

The realm of viral diagnostics has benefitted widely after the discovery of nucleic acid-based techniques due to their higher sensitivity and specificity. There are three steps primarily revolving around these methods which are isolation, amplification and analysis of nucleic acid. One of the most common nucleic acid-based detection methods for plant pathogens is Polymerase Chain Reaction (PCR) method. Currently, PCR is the basis for all diagnosis methods and is being coupled with other detection methods (López et al. 2009). While conventional PCR excels at detecting DNA viruses, the majority of stone fruit viruses possess RNA genomes. To overcome this limitation, the PCR method was adapted by incorporating an enzyme called reverse transcriptase. This enzyme enables the conversion of the viral RNA into complementary DNA (cDNA), allowing for subsequent amplification using standard PCR steps. This variation is known as Reverse Transcription PCR (RT-PCR). Multiple viruses like prune dwarf virus (PDV), cherry green ring mottle virus (CGRMV), and cherry virus A (CVA) have been detected using RT-PCR (Rouag et al. 2008; Wani et al. 2024). When the concentration of viral particles in a sample is extremely low (minimal titer), standard PCR may not be sensitive enough for detection. In this case, Nested PCR offers increased sensitivity to amplify even minute amounts of target viral DNA.

Stone fruit viruses like plum bark necrosis and stem pitting associated virus (PBNSPaV), prunus necrotic leaf spot virus (PNRSV), and plum pox virus (PPV)(Adkar-Purushothama et al. 2011) have been successfully detected using this method. Multiplex PCR adaptation comes in when multiple targets need to be detected in one go to reduce time. Using this variant, reports have been made on the detection of prune dwarf virus (PDV), apple mosaic virus (ApMV), plum pox virus (PPV), apple chlorotic leafspot virus (ACLSV), etc. (Yardimci and Çulal-Klllç 2011). Monitoring of amplification products and precise quantification of PCR products can be done using real-time PCR (qPCR). Viruses like cherry rasp leaf virus (CRLV), cherry green ring mottle virus (CGRMV), apple mosaic virus (ApMV) (Beaver-Kanuya and Harper 2020), apple chlorotic leaf spot virus (ACLSV) (Osman et al. 2017), apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV) (Beaver-Kanuya and Harper 2020), and cherry necrotic rusty mottle virus (CNRMV) (Osman, Rwahnih and Rowhani 2017) were detected using qPCR. Loop-mediated isothermal amplification (LAMP) detection is another nucleic-acid based method which uses uniform temperature and is much faster than other methods used for detection of viruses (Notomi et al. 2000). Several stone fruit viruses have been detected using LAMP method, some of which are apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), prunus necrotic ringspot virus (PNRSV) (Lu et al. 2018), etc.

Most of these methods require complex apparatus and is time consuming, hence vigorous efforts are put in to develop new and improved approaches to counter the drawbacks of the more conventional techniques. The more recent advancements in the realm of viral detection and management are listed below.

CRISPR-cas technology

CRISPR-Cas systems, or clustered regularly interspaced short palindromic repeats, are currently the target of intense study in Plant virology and allied fields. According to relative genomic analyses, CRISPR and Cas proteins form a type of an acquired immunity system to defend prokaryotic cells from invading viruses as well as plasmids, similar to that of RNA interference (RNAi) found in eukaryotes (Kotagama et al. 2019). CRISPR has a few common properties, according to comparative genomics studies: (i) found in intergenic areas. (ii) possess numerous short direct repeats with less sequence variation, (iii) have non-conserved sequences interspersed with the repeats, and (iv) have a common leader sequence of several hundred base pairs on one side of the repeat cluster. CRISPR-Cas consists of two parts which are a guide RNA (gRNA) and a CRISPR-associated endonuclease. The gRNA is specific to the sequence of the target DNA. The gRNA and Cas are joined in CRISPR research, resulting in a ribonucleoprotein (RNP) complex. In nature, transactivating CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA) combine to form gRNA. crRNA binds to the DNA target sequence and is 18–20 base pairs long. TracrRNA serves as a platform for the crRNA-Cas9 interaction to occur. The gRNA molecule is a duplex one in nature, with crRNA and tracrRNA annealed together. gRNA can be made synthetically by joining these two molecules using a linker loop (Qi et al. 2013). Cas is a protein that cuts the target DNA while gRNA directs it to the desired location (Gasiunas et al. 2012). CRISPR-Cas technology has the capacity to be used to engineer plants for viral resistance. Based on the Cas protein structure and function, there are two classes of CRISPR/Cas systems with types I, III, and IV in Class 1 system and type II, V, and VI in Class 2 system (Makarova et al. 2020). (Makarova et al. 2020). CRISPR nucleases, like Cas12 and Cas13, are revolutionizing plant pathogen detection because they can recognize and cut specific DNA or RNA sequences with high precision. This allows for rapid, sensitive, and accurate identification of viruses or other pathogens in plants, often even in the field without needing complex lab equipment.

Several advanced diagnostic systems based on CRISPR-Cas technology are increasingly employed to detect plant pathogens, microorganisms, and transgenes with high sensitivity and specificity. These systems leverage the collateral cleavage activity of CRISPR-associated nucleases—such as Cas12 and Cas13—to recognize and cleave specific nucleic acid sequences from pathogens, enabling rapid, accurate, and often field-deployable detection methods (Gootenberg et al. 2017). These platforms use the unique abilities of Cas proteins to precisely detect target nucleic acids.

The Broad Institute of MIT and Harvard developed SHERLOCK (Specific High-sensitivity Enzymatic Reporter UnLOCKing), which employs Cas13a for RNA detection and Cas12a for DNA detection. Its flexibility stems from the ability to simultaneously target multiple RNA or DNA sequences. SHERLOCK operates on the principle of collateral cleavage, where the Cas enzymes not only bind specifically to their target nucleic acids but also activate cleavage of reporter molecules, producing a detectable signal such as fluorescence. The upgraded SHERLOCKv2 system can detect up to four different nucleic acid targets in a single reaction by utilizing multiple Cas13 and Cas12 enzymes. Sensitivity is further enhanced by incorporating the Csm6 enzyme, which improves detection sensitivity by approximately 3.5-fold (Kazlauskiene et al., 2017). SHERLOCK has been extensively applied to detect various human pathogens, and its use in plant virus detection is still emerging. Notably, a recent study demonstrated the platform's capability to detect pospiviroids—plant pathogens of quarantine significance—with high specificity and sensitivity, showcasing its potential for plant pathogen diagnostics. The field of CRISPR-based diagnostics for plant pathogens continues to rapidly evolve, broadening the scope of detectable pathogens using systems like SHERLOCK.

DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) is an innovative diagnostic platform that utilizes the CRISPR-Cas12a system for detecting specific DNA sequences. In this method, the Cas12a enzyme is guided by a CRISPR RNA (crRNA) to precisely bind to a target double-stranded DNA (dsDNA) sequence. Upon recognition of the target, Cas12a exhibits collateral cleavage activity, indiscriminately cutting nearby single-stranded DNA reporter molecules, which generates a detectable signal—often fluorescence—allowing for rapid and sensitive identification of the pathogen. With its exceptional specificity and sensitivity, DETECTR is a powerful tool for fast and precise pathogen detection (Jolany Vangah et al., 2020). One study demonstrated the use of a CRISPR-Cas12a system combined with recombinase polymerase amplification (RPA) to detect Tomato Yellow Leaf Curl Virus (TYLCV). This integrated approach enabled rapid and highly sensitive detection of the virus, highlighting the significant potential of CRISPR-based diagnostics for effective management of plant diseases (Anbazhagan et al. 2024).

Besides the regulations concerning gene editing technology, the identification of PPV resistant genes from a cluster of meprin and TRAF-C homolog domain (MATHd) containing genes present in apricot trees can be facilitated for resistance breeding by marker assisted selection and assist in the gene editing approaches for PPV resistance in other Prunus species (Zuriaga et al. 2018). CRISPR-based detection methods offer a highly sensitive, rapid, and precise approach for identifying stone fruit viruses, promising to revolutionize disease management and safeguard crop health.

High throughput sequencing (HTS) technologies

High throughput sequencing (HTS), also known as next-generation sequencing (NGS), is revolutionizing plant virus diagnostics by efficiently generating vast datasets (Villamor et al. 2019). This technology integrates unique sequencing chemistries, bioinformatics tools, and diverse sequencing platforms, enabling parallel sequencing of multiple DNA or RNA strands, including entire genomes, in a short timeframe (Shendure et al. 2004; Mansoor et al. 2025). Next-generation sequencing methods have gained prominence for their role in identifying, detecting, sequencing, and uncovering various RNA and DNA viruses, as well as viroids (Adams et al. 2009; Al Rwahnih et al. 2009; Kreuze et al. 2009).These methods are also being employed for the analysis of genome diversity and evolution, strain differentiation, biogenesis, characterisation, and epidemiological studies of pathogens (Hadidi et al. 2016). NGSbased techniques follow a common methodology of extraction of nucleic acid, and the fragmentation of DNAfor library preparation (Mehetre et al. 2021). Besides their ability to determine millions of sequences in a single experiment, NGSis also utilised for comparison of these sequences with the host genome to identify genes that may be tuned due to viral infection (Wang and Smith 2016; Nabi et al. 2024). Progress in next-generation sequencing (NGS) technologies has yielded benefits across various biological disciplines, with metagenomics emerging as a notable application for virus detection. Metagenomics involves the investigation of total RNA or DNA extracted from a heterogeneous mixture of organisms (Riesenfeld et al. 2004). In this approach, nucleic acids are extracted from plant samples, followed by sequencing using NGS platforms, and subsequent identification facilitated by bioinformatics tools (Mehetre et al. 2021). Viral metagenomics is categorised under first generation sequencing methods and is being widely implemented for identification of novel viruses as they do not need any pre-requisite information on the virus or the virus-specific primers. But in the case of unidentified viruses, it becomes challenging because of the lack of reference sequences (Xiao et al. 2019).The development of these technologies led up to different NGSplatforms which were found to be more sensitive and efficient showing higher potential than traditional approaches for plant viral disease diagnostics. The most common second generation sequencing platforms are HiSeq, MiSeq, and NextSeq by Illumina, SOLiD byABI, GS FLX by Roche diagnostics, Ion Torrent PGM, S5, BGISeq-500, and Proton (Barzon et al. 2013, Lipkin and Firth 2013). Illumina HiSeq platform has been used to successfully detect prunus necrotic ringspot virus (PNRSV) in china rose plant (Kinoti et al. 2017; Loconsole et al. 2012), peach virus D in both peach and prunus (Xu et al. 2019), cherry virus A (CVA)(Baráth et al. 2018), and prunus necrotic ringspot virus (PNRSV), American plum line pattern virus (APLPV), apple chlorotic leaf spot virus (ACLSV), hop stunt viroid (HSVd); little cherry virus 1 (LChV-1) and plum bark necrosis stem pitting-associated virus (PBNSPaV) in Plum from India (Khan et al. 2024) etc. Illumina MiSeq has been used to identify viruses like apricot latent virus, apricot vein clearing associated virus (AVCaV), nectarine stem pitting- associated virus (NSPaV), apple mosaic virus (AMV), prunus necrotic ringspot virus (PNRSV), american plum line pattern virus (APLPV), prune dwarf virus (PDV) in prunus (Kinoti et al. 2017), etc. Ion torrent detected Little cherry virus 1 from sweet cherry (LChV1) (Katsiani et al. 2018), etc. Further advancement in this technology led to a method called the single- molecule sequencing techniques which generates comparatively longer reads from DNAand RNAsamples with swifter runtime, real time analysis and minimal input template (Lavezzo et al. 2016). Several platforms incorporating third-generation sequencing capabilities include the MinION nanopore technology from Oxford Nanopore Technologies, the HelicosTM Genetic Analysis System by SeqLL, and Single Molecule Real-Time (SMRT) sequencing offered by Pacific Biosciences (Ambardar et al. 2018). While the SMRT platform has been widely employed for detecting viral and bacterial pathogens in human infections, its utilization in plant virology remains unreported (Maliogka et al. 2018). The application of Oxford MinION is comparatively higher because of its portability and simplicity. This has been successfully used to process polyadenylated RNA without the synthesis of cDNA and hence supply strand specific data (Garalde et al. 2018). MinION nanopore sequencing is being used for the diagnosis and detection of various plant viruses. To sequence meta transcriptome of plant or insect tissue infected with plum pox virus (PPV), MinION nanopore sequencing technology was used coupled with whole transcriptome amplification (WTA)(Bronzato Badial et al. 2018). These third-generation sequencing technologies have immense potential to create evolutionary impact on the detection and diagnosis of plant viruses for the prompt detection of pathogens before disease severity due to its high efficiency, portability, rapid run time, enhanced read lengths, and reasonable cost of equipment. A list of viruses recently detected using NGS is listed in Table 2.

Table 2.

Novel virus-host associations revealed by RNA-Seq

Host plant	Technology type	Platform	Target virus type	Novel Virus detection capacity	Speed	New associated virus	References
Sweet cherry (Prunus avium)	HTS of RNA	Illumina HiSeq 2000	RNA viruses of Sweet cherry	Yes	High	Rusty Mottle-associated virus, Twisted Leaf-associated virus, Prunus virus F	(Villamor et al. 2016)
Sweet cherry	HTS of RNA	HiSeq 2500 sequencing	Fabavirus of Sweet cherry	Yes	High	Cherry virus F	(Koloniuk et al. 2018)
Peach	Metagenomic analysis of RNA	Illumina HiSeq 2000	RNA viruses of Peach	Yes	High	Nectarine stem pitting-associated virus	(Bag et al. 2015)
	HTS of RNA	Illumina HiSeq 2000	RNA viruses of peach	Yes	High	Nectarine virus M	(Villamor et al. 2016)
	HTS of RNA	Illumina HiSeq 2000	Small RNA sequencing of Peach viruses	Yes	High	Peach leaf pitting-associated virus	(He et al. 2017)
	HTS of RNA	Illumina HiSeq 2000	RNA sequencing of Peach viruses	Yes	High	Peach virus D	(Igori et al. 2017)
Prunus	HTS of RNA	Roche 454 Pyrosequencing	Prunus viruses	High	High	Prunus virus T	(Marais et al. 2015)
	HTS of RNA	Illumina HiScan SQ apparatus	Apricot viruses	High	High	Vein-clearing associated virus	(Elbeaino et al. 2014)

Viral metagenomics

Metagenomics utilizes nucleic acid sequencing to analyse the collective variety of viruses present within an environmental sample. In this approach, researchers directly sequence total nucleic acid extracted from infected plants through genome sequencing (NGS) platforms, followed by bioinformatic analysis to identify the viral sequences. Unlike analyzing single samples through PCR or NGS, metagenomic studies involve pooling of nucleic acids extracted from multiple viral infected plant samples within a specific area. This nucleic acid pooling analysis reveals the true diversity of viruses hiding in that environment, even those present at low levels that might be missed otherwise. Meta transcriptome analysis using RNA-Seq, which involves sequencing of all the RNA molecules within a sample, is a powerful tool for studying viruses in their natural hosts. This technique is particularly effective for single-stranded RNA viruses, the most common type of plant virus, as their RNA can be directly detected and sequenced. By analyzing the viral RNA sequences in a meta transcriptome, diversity of viruses infecting their natural or entirely new hosts can be determined. Plant pathologists leverage metagenomics for identifying potential and entirely new viruses that went unidentified previously, and unveiling the viruses that have jumped from their natural host species to new host plants (MacDiarmid et al. 2013). Few of such viruses that have recently identified to infect their non-host stone fruit crops were a new peach virus named Peach virus 1 (PeV1) infecting peach (Ghorani et al. 2024), Apple Hop stunt viroid (HSVd) and apple chlorotic leaf spot virus (ACLSV) infecting plum (Jo et al. 2020). Plant viral metagenomic studies generally utilizes either of the following classes of nucleic acids: (i) Virion-associated nucleic acids (VANA) from a purified virus, (ii) total DNA or RNA purified from virus, (iii) dsRNAs (double-stranded RNA), and (iv) small interfering RNAs (Virus derived siRNAs). Details on the use of forementioned nucleic acid for a metagenomic investigation was reviewed (Roossinck et al. 2015). Following metagenome sequencing, a multi-step process involving quality control analysis, filtering, de novo assembly, and taxonomic assignment of sequence reads is employed to identify viral contigs.

Next-generation sequencing (NGS), particularly siRNA sequencing, has transformed viral detection in crop plants. Several investigations proved the viability of utilizing siRNA sequencing to detect a wide range of known and unknown viruses and viroids in plants, including pooled samples, demonstrating that a substantial proportion of examined plants were infected (Verdin et al. 2017). These findings highlight the huge uncharted domain of plant viromes, as well as the potential for siRNA sequencing to be a critical tool for quarantine measures and future study into both symptomatic and asymptomatic plant populations.

RNA-seq

Traditional methods for virus detection often target specific known viruses, However, RNA-seq offers an unbiased approach. It analyses all RNA molecules present in a sample, enables to identify both known and unknown viruses infecting host plants. This is crucial because new viruses can emerge and cause significant damage before they are even identified. Thus, RNA sequencing (RNA-seq) turned up as a powerful tool for detection of plant viruses, offering a two-pronged approach. It allows to uncover and identify novel plant viruses by detecting all types of RNA viruses present in the infected sample and also reveals the expression of host plant genes in response to viral infection, allowing for parallel collection of host plant transcriptome data. rRNA removal is critical for accurate RNA-seq data when detecting viruses using RNA-seq. Since rRNA doesn't provide understanding into gene expression, its presence can obscure the signals from the mRNA of interest. Removing rRNA through an appropriate method from total RNA ensures a clear picture of the transcriptome and allows to focus on the most relevant RNA molecules (Nagano et al. 2015; Mansoor et al. 2024). While RNA-Seq with oligo-dT bead purification excels at detecting viruses with polyadenylated (poly(A)) tails, it has limited effectiveness for those viruses lacking them. However, the rRNA selective depression method within RNA-Seq offers a powerful solution. This approach bypasses the poly(A) requirement, enabling comprehensive detection of a wider range of viral genomes, including those without poly(A) tails (Nagano, Honjo, Mihara, Sato and Kudoh 2015). This approach has led to the identification of several putative viruses infecting stone fruit crops, jumping the species barrier and establishing themselves in new hosts. Table 2 represents some new viruses detected in stone fruits through RNA-Seq which are unreported earlier.

Conclusion and future prospects

The emergence of new viruses affecting fruit crops continues to be a global threat for the entire fruit industry. Viruses have an unusual nature with a deceptively simple topology. Plant viruses weaken the host system and affect yield and yield-related traits ultimately degrading the quality. Due to their nature to influence the growth and development related factors in the plant, the economic loss is peak affecting both growers and consumers. Early detection and diagnosis to generate early results for analysing and preventing disease are key for management of these viruses. Besides these, host-virus relationship must be better understood for the effective management. Even with advancements made in all aspects, cultural methods, molecular-based methods, and immunological methods have multiple shortcomings as most of them are time consuming, requires complex apparatus, and expensive chemicals.

Plant virologists are now being encouraged to utilise the advanced technologies like CRISPR-Cas and NGS based techniques for analysis and diagnosis. The approach of using RNAi mechanism for further inhibiting viral protein synthesis or preventing the incidence of virus infection has shown great potential for countering these viruses. Topical application of dsRNA is a non-transgenic, environment-friendly method that can effectively replace genetic engineering of the susceptible plants. The application of these techniques is not just confined to diagnosis and detection but in detailed research studies as well. Though significant progress has been made in the technological advancements related to detection and diagnosis of viruses, consistent innovation is required for improving diagnostic techniques to aid early identification and containment of these diseases. Notable advancements have been made in plant viral disease diagnosis but inexpensive and simple techniques are still under developed. Infections by multiple viruses also remain a major barrier for most of the developed technologies. Accurate choosing of target site for molecular detection techniques is complex and requires years of expertise. Novel detection techniques with qualities like affordability, simplicity, rapidity, specificity, user-friendly, and extended shelf life must be encouraged. Further, bioinformatic tools like NGS methods are showing high potential as the next big thing in plant disease diagnosis and detection, particularly on viruses.

Author contributions

Conceptualization, S.N., and S.W.; Writing- Original draft preparation, S.W., N.I., S.M and D.M.; Writing- Review & Editing, S.N., M.S., A.H. S.M and S.R.; Supervision, S.N., M.S., S.M and A.H. All authors have read and agreed to the published version of the manuscript.

Funding

Not applicable.

Data availability

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors affirm that there were no financial or commercial ties that might be interpreted as a potential conflict of interest throughout the research.