Introduction

Papaya (Carica papaya L.), often referred to as the "Wonder Fruit of the Tropics," is a fruit crop with significant economic value that is cultivated extensively in tropical and subtropical areas across the globe. In India, papaya is cultivated in an area of 148.81 thousand hectares with a production of 5341.83 million tonnes and productivity 35.90 tonnes per hectare [1]. In Tamil Nadu, India it is grown in an area of 3.21 thousand hectares, with a production of 0.219 million tonnes and productivity of 68.40 tonnes per hectare [2]. Numerous pests and diseases cause significant losses in papaya production, with papaya ring spot disease being the most destructive. The rapid spread of the virus allows it to infect upto 100% plants, causing a serious reduction in yield loss. In an area that is severely affected, farmers have to stop growing papaya due to the devastating effects of the infection [3]. In light of the yield loss attributed to the disease, various management strategies have been implemented to combat the virus. While many conventional approaches employed years ago persist today, their outcomes in controlling the virus have proven less effective. This review focusses on the importance of the papaya ringspot virus (PRSV), symptomatology, and transmission as well as different molecular techniques including breeding approaches, transgenic and non-transgenic strategies employed in combating PRSV in papaya.

Genome organisation

PRSV belongs to the genus Potyvirus, the largest group of the plant virus under the family Potyviridae. The virion of PRSV is a non-enveloped, monopartite, filamentous and flexuous rod-shaped, single-stranded positive RNA genome measuring 760–800 × 12 nm. The genome of PRSV is about 10,326 nucleotides with 5’ VPg and 3’ poly-A tail encapsidated by viral coat protein (CP). A single open reading frame (ORF) beginning at nucleotide 86 and closing at nucleotide 10,120 encodes a polyprotein with 3344 amino acids (aa) (Fig. 1). The typical virus particle contains 5.5% nucleic acid and 94.5% protein. The thermal activation point (TIP), the longevity in vitro (LIV) and purified virion buoyant density in cesium chloride are 54–60 °C, 0.3 days and 1.32 g cm−3 respectively [4]. The proteolytic cleavage of this protein results in 8–9 smaller proteins with various functions viz., P1 (63 kDa), helper component-protease (HC-Pro, 52 kDa), P3 (46 kDa), 6K1 (6 kDa), cylindrical inclusion protein (CI, 72 kDa), 6K2 (6 kDa), nuclear inclusion a (NIa, 48 kDa), nuclear inclusion b (NIb, 59 kDa) and coat protein (CP, 35 kDa) [5].

Fig. 1
figure 1

Genome organisation of papaya ringspot virus

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Among these P1, HC-Pro and NIa-Pro act as virus-encoded endoproteases. P1 shared 67–84% aa sequence identity and is the most variable region among other PRSV isolates while other segments like HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa, NIb and CP shared 91–100% aa similarity [6]. P1 not only acts as a proteinase but is also responsible for cell-to-cell and systemic movement of the virus in infected plants. HC-Pro is a non-virion versatile protein involved in vector transmission, proteinase, pathogenicity, long distance movement and suppression of post-transcriptional gene silencing (PTGS)—a crucial defense mechanism employed by plants against viral infections [7]. Notably, HC-Pro is a highly effective suppressor of RNA silencing, a process that plays a crucial role in the regulation of gene expression and development in plants. This suppression of RNA silencing can have significant implications, as it can disrupt the microRNA-mediated developmental pathways in the host plant, thereby contributing to the establishment and proliferation of the heterologous virus[8].

The P3 function is unknown, but it is considered to play a possible role in replication [9]. 6K1 is involved in RNA replication, regulation, and inhibition of NIa nuclear translocation [4]. CI protein is involved in NTPase, NTP binding, RNA binding and RNA helicase activity, membrane attachment, cell-to-cell movement, induces amorphous and cylindrical inclusion body in the host cell cytoplasm[5]; 6K2 has the same function with 6K1 [4]. NIa contains two domains namely N-terminal viral protein genome-linked (VPg) and C-terminal protease domain. VPg acts as a primer for RNA synthesis initiators [5]. NIb acts as an RNA-dependent RNA polymerase [9] Both NIa and NIb are involved in the production of nuclear inclusion body [10]. CP involves encapsidation of viral RNA, vector transmission, pathogenicity, and cell-to-cell movement [11]. The 5’ untranslatable region (UTR region) of the PRSV genome of 85 bp is similar to other Potyvirus and is a rich source of A and U residues indicating that this region serves a shared functional role but is 63- 121 bases shorter [12].

Symptomatology and transmission

PRSV has two predominant pathotypes: the papaya (P) type infects Caricaceae, Cucurbitaceae and Chenopodiaceae, whereas the watermelon (W) type infects Cucurbitaceae and Chenopodiaceae. Typical symptoms produced by PRSV P type on papaya (Fig. 2) include leaf mosaic and chlorosis, flower abortion, stunting of infected plants, shoe string-like symptoms on young leaves, leaves malformation and puckering, moist oily-saturated streaks on the petiole and upper portion of the trunk [10]. Infection during the early vegetative stages results in stunting of plants, bunchy top, and never bear fruit, which would lead to complete yield loss. On the other hand, infection during the reproductive stages results in yellowing of the entire leaf and development of ring spot on fruits resulting in 85.0–90.0% yield loss [13]. As a result, fruit sugar levels drop by 50% or more and fruit production is reduced drastically [10]. PRSV is transmitted to healthy plants through sap of the infected plants [14], seeds (23.40%) as well as by several species of aphids viz., Myzus persicae (93.33%), Aphis gossypii (90.00%) and Aphis craccivora (83.33%) respectively in a non-persistent manner [15]. Hence, the disease severity in farmer field is very high because of its easily transmissible nature. Therefore, proper and well-planned management strategies are required to prevent the spread of the virus.

Fig. 2
figure 2

Various symptoms of papaya ringspot virus on papaya. a Flower abortion; b Leaf mosaic and chlorosis; c Water-soaked circular lesion. d Shoe-string symptom; e Malformation and leaf puckering; f Stunted growth of the infected plant

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Management of papaya ringspot disease

The farming community has implemented various strategies to combat the papaya ring spot disease, but none of them have yielded effective results. Reinforcing the plant's resilience against viral infection, curtailing the virus's spread, and eradicating or averting the source of infection form the basic concepts of virus control. The approaches employed to manage viral diseases can be broadly categorized into conventional methods, breeding approaches, and transgenic and non-transgenic approaches. A detailed explanation of the various PRSV control techniques and flowchart (Fig. 3) is provided below.

Fig. 3
figure 3

Overall view for the management of PRSV

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Conventional approaches

The key cultural or agronomic methods for preventing the spread of PRSV include shifting cultivation [3]; ensuring an adequate isolation distance from previously infected fields; the use of virus-free healthy seedlings; modification of the transplanting period, encircling papaya plantations with border crops to prevent aphid-vector access, destroying of infected plants, weeds and other reservoir plants; and alternate cropping system [16]. Despite efforts, these methods of controlling the virus proved unsuccessful. Rouging did not completely stop the infection, and using insecticides wasn't effective because aphids spread the virus before they were eliminated. Complete eradication of orchards is ineffective because some virus strains can survive in weeds, other perennial plants, and annual plants, especially those that are cucurbits [17]. Hence, to effectively control virus spread and maintain papaya productivity, it is imperative to look for alternative and effective control strategies.

Breeding strategies

Farmers can control plant viruses most easily and affordably by using resistant cultivars. However, it takes time to introduce resistance genes into commercial cultivars because it needs to be present in the germplasm of the target species. Before initiating the breeding process, it is crucial to confirm that the resistance is not compromised by any pre-existing virus strains, or whether the virus can quickly adapt to get past it. Many species in the genus Vasconcellea showed complete resistance to PRSV, making them an important source of promising genes which can be exploited in papaya breeding program. However, because of their genetic distance, intergeneric hybridization between Carica and Vasconcellea could not be successfully carried out, leading to infertility, abortion of developing embryos, and inadequate hybrid vigour. These outcomes have made it difficult to transfer resistance genes to papaya using traditional methods [18]. Furthermore, traditional breeding techniques between wild and cultivated papaya species cannot produce PRSV-resistant varieties due to their sexual incompatibility with each other [19].

Molecular markers, on the other hand, are employed in the breeding of papayas to identify hybrids, ascertain the genetic proximity between different genotypes, and pinpoint specific sequences associated with desirable characters that can serve as markers [20]. Molecular markers can shorten the periods needed for a breeding program by assisting in the selection of hybrids resistant to PRSV. Randomly amplified polymorphic DNA (RAPD) was employed to evaluate the genetic associations between Vasconcellea and C. papaya species as well as among papaya cultivars [21]. V. cundinamarcensis was found to have markers associated with PRSV resistance identified using a variation of this method called randomly amplified DNA fingerprint (RAF) [22]. Psilk4, a CAPS marker, has demonstrated 99% accuracy in identifying resistant genotypes when used to separate V. parviflora X V. cundinamarcensis F2 progeny for PRSV resistance [18].

Marker-assisted breeding has utilized molecular markers associated with PRSV-1 gene due to its dominant inheritance [23]. Fluorescence in situ hybridization (FISH) was used by Fabiane et al. [24] to investigate genomic and chromosomal relationships among the species of Caricaceae. The microsatellite loci of papaya released by de Oliveira et al. [25] furnished an extensive collection of genetic markers. A total of 16 primer pairs were investigated by Alamery and Drew [26] which revealed that no resistance genes were found to be associated with simple sequence repeat (SSR) markers in the segregating populations and the hybridity of F1 was confirmed by one marker, SP16. Four markers associated with PRSV resistance in V. pubescens were found by Razali and Drew [27] in an interspecific mapping population of V. pubescens and V. parviflora. One of them was a practical marker that was found in the same spot as PRSV-1 and shared similarities with genes for serine threonine protein kinase (STK) [16].

Transgenic approaches

Transgenic plants resistant to viruses can be developed using the concept of pathogen-derived resistance (PDR). In PDR, the pathogen sequence or genes are introduced into the target plant to protect it from the pathogen or closely related groups[28, 29]. An effective RNA-mediated protection requires a transgene that is highly similar to the challenging virus [5]. Gene silencing involves targeting any viral sequence to confer resistance, without the need for protein expression [30]. The transgenic papaya line developed through RNA-mediated mechanisms is mainly based on PTGS which depends on the homology sequence between the viral genome and corresponding transgenes [31]. Various transgenic papaya lines have been developed targeting the genomic regions of PRSV (Table 1). Scientists at American and Chinese universities have currently approved and developed four genetically modified papaya events. The first genetically modified papaya was produced by two events that were developed at Cornell University and the University of Hawaii. They were derived from the cultivar "Sunset" and were named 55-1 (OECD UID: CPH-CP551-8) and 63-1 (OECD UID: CPH-CP631-7). The "Huanong No.1" event, which was not registered with the OECD, was released concurrently by South China Agricultural University. X17-2 (OECD UID: UFL-X17CP-9) developed at the University of Florida is the fourth event, deregulated in the United States and registered by the EPA in 2016 [17]. However, developing PRSV-resistant transgenic plants is challenging due to differences among geographically distinct isolates [5].

Table 1 Papaya ringspot virus resistant transgenic lines developed by different countries
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Coat protein-mediated resistance

The mechanism behind CP-mediated resistance is that CP will interfere in the early stage of virus infection by preventing virus replication and movement thereby restricting the spread of viral infections within the plant. The first successful papaya transgenic line was developed by Fitch et al. [32] by incorporating PRSV HA 5-1 CP gene resistant to severe Hawaii HA strain via microprojectile bombardment into immature zygotic embryos by the constructing plasmid containing neomycin phosphotransferase II (nptII) gene [5, 14]. Transgenic papaya line 55-1 developed by triggering PTGS are highly resistant to Hawaii PRSV isolates. The resistance is due to the sequence identity between the challenge virus CP gene coding region and CP transgene [43]. Rainbow and SunUp, that have been developed through CP-mediated mechanisms, were the first transgenic fruit trees being commercialized [44]. These two varieties provide an excellent solution to Hawaii's PRSV problems [45]. However, Rainbow, a CP-hemizygous line developed by a cross between SunUp and non-transgenic Kapoho, is found to be susceptible outside Hawaii. SunUp is a CP-homozygous line of 55-1 and susceptible to isolates from Taiwan and Thailand but shows resistance toward Brazil and Jamaica isolates [46] [44]. Crossings between GM and non-GM papaya produced additional varieties with less significance in the world's production. One such is the micro-propagated "Laie Gold," a hybrid between the non-GM "Kamiya" and the "Rainbow" F2 that is distinguished by shorter trees and initially higher yields [47].

The resistance of five X17-2 progenies to the PRSV strains H1K, H1C, and H1A that naturally occur in Florida was assessed between 1999 and 2007 through pollination with non-GM varieties. Unlike non-GM plants, which showed severe disease symptoms, they were resistant to H1K and tolerant to the other two strains [17]. According to APHIS [48], Florida papaya growers may find success with hybrids created from the PRSV-resistant X17-2. Cheng et al. [33] constructed Ti binary vector pBGCP using the CP gene of the Taiwanese PRSV YK strain through Agro-mediated transformation and the lines obtained were resistant to PRSV YK as well as to three different geographical strains from Thailand, Hawaii and Mexico. However, after 18 months of the fourth field trial, symptoms of severe papaya ringspot disease appeared [49] which was later confirmed as papaya leaf distortion mosaic virus (PLDMV) [50]. It was reported that CP-mediated transgenic papaya lines which were resistant to PRSV were found to be susceptible to PLDMV. A chimeric construct containing the CP genes of PRSV and PLDMV can be used to create a transgenic papaya line that is double resistant to both viruses [14]. Thus, the development of effective PRSV-resistant plants appears to be possible with the use of CP-mediated mechanisms.

Replicase-mediated resistance

Replicase-mediated resistance can be employed by introducing replicase gene into the plant. This gene will interfere with the virus replication by triggering RNA interference (RNAi) mechanisms thereby preventing virus replication in transgenic plants. Replicase protein serves as the basis for the resistance mechanism because transgenes encode the primary structure of the protein, which is altered by mutations, resulting in the resistance phenotype. The structures of replicase genes varied amongst different genera [11]. The first demonstration of replicase gene-mediated resistance was demonstrated in Nicotiana tabacum against the TMV [51]. Chen et al. [42] developed transgenic papaya resistance to PRSV by introducing a virus replicase gene. Agrobacterium tumefaciens carrying the pRPTW vector was used to transform embryogenic calli of the cultivar 'Tai-nong-2'. Mini Ti plasmid vector pRok was constructed under the instruction of the CaMV 35S promoter and terminator nopaline synthase gene (NOS) with replicase gene fragment orienting 3’-truncated and 5’-extended. Replicase-containing transgenic papaya plants were found to be protective against PRSV, and those with mutated replicase genes exhibited a high level of resistance to the virus [52]. China deregulated ‘Huanong No.1’, a transgenic PRSV-resistant papaya variety for commercial production in 2006 [53]. This cultivar was obtained through PDR but instead of the CP gene, the replicase gene was used [5, 10]. In the first five to six years, the transgenic papaya plants with the replicase gene showed no signs of resistance loss [54]. However, during the past few years, Zhao et al. [55] reported resistance breakdown of PRSV in "Huanong No. 1."

HC-Pro-mediated resistance

HC-Pro is an important component of PRSV that helps in vector transmission, RNA silencing suppression, cell-to-cell movement, responsible for synergisms between potyviruses and unrelated viruses that leads to severe symptom production and virus accumulation in infected leaves [5, 11]. Transgenic plants with strong virus resistance are predicted to arise from the silencing of the HC-Pro gene, which will directly downregulate viral RNAs and increase host antiviral silencing activity [56]. HC-Pro is a crucial element that must be taken into account for PRSV-resistant papaya to develop on the Indian subcontinent [57]. Cheng et al. [58] developed new mutants of the PRSV YK strain by modifying the HC-Pro of PRSV YK. Among the mutants, F7I and F7I + F206L, displayed mild symptoms in papaya plants, indicating that the virus was less successful in suppressing RNA silencing in these mutants than in the wild type. Under greenhouse conditions, F7I and F7I + F206L mutants were stable, offered complete protection against the PRSV YK strain, and were found to be difficult for aphids to spread the virus.

However, papaya plants developed using PTGS through transgenic approaches encounter reduced public acceptance due to biosafety concerns. Consequently, there is a pressing need to devise alternative methods that address both aspects i.e., effective plant control and public safety. This prompts a consideration of non-transgenic methods, which can be used for the management of the virus.

Non- transgenic approaches

An alternative strategy for controlling PRSV involves non-transgenic approaches, such as cross-protection, genome editing and topical application of dsRNA. These methods harness RNAi mechanisms, serving as intrinsic plant immune responses, to facilitate the recovery of virus-infected plants. Additionally, they contribute to the control of insect vectors through gene-silencing mechanisms thereby preventing the spread of the virus. We are set to discuss the details of each method as well as shown in Fig. 4, unravelling its intricacies to provide a clear understanding of its mechanisms and practical applications.

Fig. 4
figure 4

Non-transgenic approaches for the management of PRSV

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Cross-protection

Cross protection is a mechanism in which a plant when infected systematically with a mild strain of virus prevents the infection of a severe strain of the same virus. The key feature for practical application is the availability of mild protective virus strains. However, this naturally occurring mild strain when used for practical application was not mild enough to control the virus disease. Thus, the effort of selecting mild strains from the natural population to control viral disease was shifted to artificial mutagenesis [59]. Two attenuated PRSV mutates viz., HA 5-1 and HA 6-1 were inoculated on papaya seedlings and found to be symptomless without the plant's size being reduced. But when this sap was tested in double antigen sandwich-enzyme linked immunosorbent assay (DAS-ELISA) using antiserum against PRSV, it was found to be positive indicating symptomless infection was not due to slow replication or low concentration of virus [34]. Under greenhouse conditions, the mild mutant PRSV HA 5-1 was used against severe PRSV HA to protect papaya seedlings. Prior to inoculation with severe strain PRSV HA, the seedlings were pre-infected by gently rubbing the individual leaves with mild strain PRSV HA 5-1 at five to six-leaf stages at different time intervals [60]. It was found that most of the plants were symptomless even after 3 months from the day of inoculation. However, mock-inoculation of papaya seedlings with buffer first followed by re-inoculation with PRSV HA produces severe symptoms at 15 days [34].

In vivo, construction of mild strain cDNA using the backbone of HA 5-1 expressing NIa, NIb and CP regions from TG5, severe Vietnam strain, either singly or combined revealed that, mild recombinant HA5-1/TG5-CP3′ maintained high levels of protection against homologous HA and greatly enhanced protection against heterologous TG-5. Similarly, papaya plants with HA 5-1 recombinants containing single CP3′ fragments from Taiwan YK, Vietnam ST2 severe strains, and Thailand SMK strains also greatly boost defence against the corresponding heterologous strains [61]. Chiang et al. [62] used a mild mutant of the severe strain of the PRSV, HA 5-1, as a model to study the genetic basis of attenuation. They tested the infectivity of the recombinants produced by crossing HA 5-1 with HA on papaya and Chenopodium quinoa. Infectious clones containing recombinants of mutated P1 and HC-Pro reduced infection on papaya with no obvious symptoms similar to the mild strain. Additionally, the Indian Agricultural Research Institute, Regional Station in Pune, India, has attempted to control the severe strain of PRSV isolates in Pune using a mild strain of PRSV that occurs naturally and offers 66% resistance [3]. Even though this technique of using attenuated virus strain to control economic damage of severe virus strain was effective, it has several demerits [63].

Genome editing

The development of virus-resistant plants can be accelerated using CRISPR-associated short palindromic repeats (CRISPR). These modern advances in genome editing have made it possible to engineer plants with antiviral defences that are highly effective. CRISPR/Cas9 technology, in particular, functions like a molecular cutter by rupturing specific sequences in its substrate's DNA or RNA molecules. Engaging resistance against plant viruses through CRISPR-Cas technology has shown to be a promising strategy [64]. PRSV relies mostly on host factors (eIF4E or eIF(iso)4E) to complete its life cycle by interacting viral VPg with the hosts [65, 66]. CRISPR Cas9 specifically targeted eukaryotic initiation factor 4E (elF4E), a gene located in the host genome. It is known that this gene plays a role in the potyvirus infection process. Through its interaction with the viral protein known as VPg, the elF4E gene aids in the replication of the viruses [67]. CRISPR-based genome editing for papaya breeding is now possible owing to the availability of whole-genome sequences and papaya genetic transformation technologies [68]. The transgenic plant can be developed by either editing genes of the host which are crucial for virus multiplication or by directly targeting the virus [63, 69]. The translation-promoting 3′ poly A tail of mRNA and the potyviral 5′ m7G cap structure are bound by the eIF4E complex. Further viral infection is prevented when this interaction is disrupted by either mutagenesis or silencing [70]. However, to control viral diseases in tropical fruit crops, more studies on the diverse uses of the CRISPR/Cas9 methodology are needed. Application of the CRISPR/Cas9 system has already been made to fruit crops grown in tropical and subtropical areas but studies on a few important tropical fruit crops, like pineapple, are non-existent or very rare like papaya for disease resistance [71]. Even so, CRISPR/Cas is fast emerging as a potent tool for creating non-transgenic plant varieties that are resistant to viral diseases, thereby reducing the loss of agricultural productivity and promoting food security.

Topical application of dsRNA

To combat plant diseases, biotechnologists are investigating non-transgenic strategies that can generate strong resistance to viruses. Similar to transgenic RNAi-derived resistance exogenous application of dsRNA molecules can specifically induce RNAi-derived non-transgenic virus resistance plant [72]. The first case of Potyvirus resistance to both the homologous isolate and an additional PRSV isolate was conferred by applying dsRNA molecules exogenously from one PRSV isolate. The PRSV-Tirupati isolate's dsRNA molecules from the CP and HC-Pro genes provided complete resistance to infection. Additionally, the same dsRNA molecules were extremely successful in combating PRSV-Delhi isolate on the papaya cv. Pusa Nanha, providing resistance of 94% and 81%, respectively [73]. Shen et al. [74] used PRSV CP-mediated intron-containing hairpin dsRNAs or hpRNA-CP279 expressed in Escherichia coli strain M-JM109lacY for developing non-transgenic resistance papaya by directly spraying on plant. Resistance analyses and ELISA data confirmed that, manual co-inoculation of TRIzol-extracted ihpRNA-CP279 and PRSV showed resistance throughout the test period of more than 2 months after post inoculation whereas sequential inoculation of PRSV and ihpRNA-CP279 with 1–2 days interval leads to delay of PRSV symptoms by 3 to 4 days but fail to provide complete protection against PRSV.

However, naked RNA being unstable can be easily degraded when exposed to the environment. To increase the shelf life of RNA and to facilitate efficient transportation inside the plant, nano-based carriers like clay nanosheets, chitosan, lipid-modified polyethyleneimine, carbon dots, mesoporous silica nanoparticles, carbon nanotubes, DNA nanostructures etc. can be used [75]. One such example is the use of clay nanosheets for the delivery of dsRNA. Exogenous application of clay nanosheet dsRNA increases the protection period up to 20 days following the application. This protection can be observed regardless of the viral inoculation sites suggesting that dsRNA can transport from topical application sites to other tissues [76]. Some of the dsRNA delivery methods include mechanical inoculation, spreading by brushes or pipettes, spray application, use of RNA adhesion materials, loading on nano-carrier and delivery by root soaking [77]. This method of topical application of dsRNA is a more environmentally friendly, long-term, and socially acceptable method. In addition, this method has various benefits over plants developed by tissue-culture and genome-editing techniques, including simple multiplexing, minimal risks of producing off-target effects and somaclonal variant plants, minimal metabolic load on host plants as a result of non-constitutive induction of RNAi, and a host of other benefits results [78].

Targeting insect vector

Controlling insect vectors is one of the most important strategies for managing the dissemination of virus diseases. The use of insecticides in large amounts not only has a negative impact on the environment but also harms the beneficial insects. Nowadays, with the advancement of molecular sciences, gene silencing mechanisms can be employed as novel pest management strategies. For silencing plant insect vectors, there are two approaches: the first approach is silencing the target gene involved in the synthesis of essential cellular components, insect hormone homeostasis, energy metabolism, chitin metabolism, digestive metabolisms, detoxification, immunity, insecticide resistance, and other processes [79] and the second approach interferes with virus transmission pathway. Various techniques can be used to introduce RNAi inside the vector body. The RNAi experiment's success is contingent upon the target gene, the dsRNA's length range, and the mode of delivery. Target gene silencing must significantly reduce the insect's fitness or result in its death for RNAi to be considered effective [80].

Different hemipteran species, including aphids, have been subjected to RNAi and it been noted that it works best when directed against the genes of the salivary glands and midgut. For dsRNA in hemipterans, three main delivery methods have been used: feeding transgenic plants, oral delivery, and microinjection. Out of all these, microinjection is the least used because it is relatively more expensive and does not allow for manipulation because the tiny insect's physical damage triggers an immune response. Hemipterans have demonstrated efficacy when fed an oral diet containing dsRNA [81]. It is a less complicated method of delivering dsRNA and does not harm the insect. However, it is less effective due to low silencing efficiency and unable to generate large amounts of dsRNA. Furthermore, the creation of transgenic plants that produce dsRNA targeting particular insect vital genes is another method that has been applied to hemipterans. The main concept to bear in mind when using this technique is the expression of enough dsRNA to cause the insects to exhibit lethal phenotypes. The dsRNA should be engineered to be extremely specific to the target gene and to have a distinct binding mechanism. This is a very important step because it should make sure that there is no chance of non-specific targeting of other genes and organisms [82].

Another method is the topical application of dsRNA or siRNA formulation. Despite the cuticle barrier of insects, dsRNA or siRNA has been shown to enter and cause death to numerous insects. However, the ability of dsRNA to enter inside the insect body is quite limited due to the thick cuticle. Nanoparticles such as liposomes, chitosan and cationic dendrimers improve shelf-life, and stability and facilitate better uptake across the cell membranes [83]. BioClay loaded with dsRNA when applied exogenously to the host protects from the virus when applied five days before exposure to aphids Myzus persicae [84]. Building on the above ideas, we can formulate powerful management strategies to control aphid vectors responsible for PRSV dissemination through the application of RNAi gene silencing mechanisms.

Future perspective and conclusion

The prevalence of PRSV poses a significant challenge to global papaya production. Traditional methods and breeding approaches have proven ineffective in the control of PRSV, necessitating the evolution of transgenic papaya varieties using gene technology. However, instances of transgenic resistance breakdown, as observed in "Rainbow" and "SunUp," raise concerns. Despite this, the adoption of PRSV-resistant transgenic papaya remains slow, influenced by factors such as societal acceptance, environmental concerns and adherence to biosafety laws. Recent research indicates that PRSV-resistant transgenic papaya presents no discernible threat. An alternative approach involves controlling the spread of PRSV between plants by targeting insect vectors through gene silencing mechanisms, such as RNAi. Additionally, emerging gene editing technologies like CRISPR/Cas9 offer a non-transgenic means of developing PRSV-resistant plants. Another avenue involves the topical application of dsRNA or siRNA, providing non-transgenic alternatives for creating PRSV-resistant plants.

With continued research into non-transgenic alternatives like RNAi applications through foliar application, along with advancements in gene technology, it is possible that in the near future, more robust and extensively used papaya varieties resistant to PRSV will be introduced, promoting safe and sustainable papaya production worldwide.