Thrips tabaci, which transmits Tomato spotted wilt virus (TSWV) in a persistent and propagative manner, varies greatly in its ability to transmit different isolates of TSWV. Similarly, TSWV isolates are transmitted at different efficiencies by different populations of T. tabaci. This study characterizes differences in virus titers in the vector among TSWV isolate-T. tabaci isoline pairings in relation to differences in transmission rates, and demonstrates that although transmission rates were higher for sympatric than allopatric TSWV isolate-T. tabaci isoline pairings, virus titers in the thrips vector were significantly lower in the sympatric pairings. Results further demonstrate that TSWV titers in the vector were unrelated to virus titers in the leaf tissue from which they acquired the virus and provide evidence for the importance of specific vector-virus interactions and local adaptation in determining transmission efficiency of TSWV by T. tabaci.Plant viruses rely on insect vectors for transmission among plant hosts, but many of the specifics of virus-vector interactions are not fully understood.  Most plant viruses are dependent on insect vectors for plant-to-plant transmission. Specific interactions with their insect vectors are required for the viruses to move to new hosts but little is known about how these interactions impact the efficiency of transmission. This study examines the relationship between virus titer in the vector and transmission efficiency across multiple isolates of Tomato spotted wilt virus (TSWV)  and isolines of its thrips vector, Thrips tabaci, originating from multiple geographical locations and host plants. Specifically, we examine differences in transmission rates among TSWV isolate-T. tabaci isoline pairings in relationship to differences in virus titers in the vector and whether these relationships differ between sympatric and allopatric pairings of TSWV isolates and T. tabaci isolines.Modern agricultural practices and globalization of trade have contributed to a surge of emerging plant viruses that are responsible for billions of dollars in annual crop losses. The expansion of agricultural land alters stable relationships between viruses, insects, and their natural plant hosts; providing opportunities for viruses and vectors to exploit widely available cultivated hosts4. For persistently transmitted viruses, successful transmission requires that the viruses traverse anatomical barriers in their vectors. After ingestion, the virus must move from the gut lumen, across other tissues or through hemolymph, and into the salivary glands from which the virus can be transferred into plants during insect feeding5. Transmission and transmission efficiency of viruses are ultimately determined by the number of virions that accumulate in the appropriate salivary compartments after circulation and/or replication within the vector.Vector competence reflects the success of the virus in overcoming intrinsic, physiological, microbiota, and immunity barriers, and can be governed by genetic interactions between vector and viral genotypes that are subject to influence by extrinsic factors, including host plants and environmental conditions10. Circulative, non-propagative viruses in the families Luteoviridae, Geminiviridae, and Nanoviridae are transmitted by aphids, whiteflies or leafhoppers. Because these viruses do not replicate in their vectors, higher virus titers in plants10 and/or longer feeding periods11 have been shown to increase the amount of virus acquired by the vector and increase transmission12. In persistent-propagative viruses, less is known about how virus titers in source plants and in the insect vector relate to transmission efficiency. There is evidence in the genus Tenuivirus that virus titer in source plants positively correlates with transmission efficiency by planthopper vectors7, but this relationship was not consistent among isolates of Maize stripe virus (MStV)6. Virus titer is also believed to be a determinant for transmission of TSWV because higher titers of TSWV in adult thrips have been related to higher transmission frequencies in the efficient vector F. occidentalis; however, differences in transmission among thrips species varying in the efficiency with which they transmit different tospoviruses are not fully explained by virus titers in the vector16.Several studies on vector taxa exhibiting different modes of transmission have shown positive correlations between viral titer within the source plant or insect vector and the efficiency of transmission of plant viruses17 examining transmission of 89 distinct pairings between TSWV isolates and Thrips tabaci isolines showed a significant effect of virus isolate, thrips isoline, and their interaction on transmission efficiency. Although transmission rates ranged from 0–55% across all isolate by isoline pairings, the ability of a single T. tabaci-isoline to transmit multiple virus isolates varied up to 18-fold, and the transmission rates of each TSWV isolate by multiple T. tabaci isolines varied up to 45-fold. In addition, significantly higher transmission rates were observed among sympatric (originate from the same location) TSWV isolate-T. tabaci isoline pairings than allopatric pairings (originate from different locations), suggesting local adaptation between virus and vector resulting from coevolution in which local virus has greater infectivity than foreign virus on local vectors18. The purpose of this study was to examine whether or not the observed variation in transmission frequency was influenced by differences in virus titer within the vector. Both the leaf tissue and thrips used in the studies of Jacobson and Kennedy17 were flash frozen in liquid nitrogen immediately following their acquisition and inoculation access periods, respectively, and stored at −80 °C until used in this study. We used a subset of the TSWV isolate and T. tabaci isoline pairings studied by Jacobson and Kennedy17 to determine if TSWV titers in adult T. tabaci varied among the TSWV isolate and T. tabaci isoline pairings. We also examined whether differences in transmission rates were related to variation in virus titers in the leaf tissue from which the thrips acquired the virus and in the thrips vector.Although the aforementioned studies document a relationship between virus titer and transmission frequency for certain virus-vector pairings, they focused on thrips from established laboratory colonies. They do not address the relationship between virus titer in the vector and inter-population variation in transmission within a single vector species or variation among isolates of TSWV. A previous study by Jacobson and KennedyThrips tabaci and TSWV isolates included in our study were subsamples of those tested by Jacobson and Kennedy17 in their characterization of differences in transmission efficiency among different pairings of T. tabaci isolines and TSWV isolates, both of which were obtained from multiple locations and host plants in North Carolina. The T. tabaci individuals studied were from clonal isolines of T. tabaci that were established from thelytokous females  collected at each location to minimize genetic variation within each of the isolines. Transmission efficiency of each TSWV isolate by each T. tabaci isoline was then characterized. Details of collection, establishment of clonal thrips isolines, TSWV isolates, transmission experiments, and results from transmission assays to characterize each TSWV isolate-isoline pairing are described in Jacobson and Kennedy17. They classified individual thrips as transmitting or non-transmitting based on a DAS-ELISA test of the leaf discs on which viruliferous thrips were allowed to feed during the inoculation access period (these leaf discs were no longer available for the experiments reported here). Samples of transmitting thrips, non-transmitting thrips, and infected leaf tissue used for acquisition of TSWV from each isolate-isoline pairing were flash frozen in liquid nitrogen and stored at −80 °C until used in the experiments reported here. A subset consisting of 12 of the 89 TSWV isolate-T. tabaci isoline pairings for which transmission efficiencies were reported by Jacobson and Kennedy17 were chosen for the experiments reported here; they were representative of the range in transmission efficiencies observed among the 89 TSWV isolate-T. tabaci isoline pairings and included both sympatric and allopatric pairings17  were utilized to design additional primer pairs , P = 0.9853] , P = 0.0820]  replaced the interaction term in the ANOVA model. Titers were measured in 39 individual thrips representing sympatric TSWV isolate-T. tabaci isoline pairings and 66 individual thrips representing allopatric TSWV isolate-T. tabaci isoline pairings. In this analysis, virus isolate  and T. tabaci isoline  main effects on virus titers per thrips were both significant. In addition, the effect of location was significant; mean virus titers of adult thrips were significantly lower for sympatric than allopatric virus isolate-thrips isoline pairings  , 01] Fig. , whereas, 93, 01]001 Fig. ], indicaT. tabaci included in our study, 42 percent had transmitted TSWV to leaf discs and 58 percent had not transmitted. TSWV was detected in all 105 thrips regardless of whether they transmitted the virus, and the mean virus titer per thrips was significantly higher in transmitting than non-transmitting thrips   was included as a main effect. Jacobson and Kennedy17, in their prior study involving a larger number of TSWV isolate-T. tabaci isoline pairings, found that transmission was higher in sympatric TSWV isolate-T. tabaci isoline pairs than allopatric pairs; a result indicative of local adaptation20. Within the subset of TSWV isolate-T. tabaci isoline pairings that were included in our study, the mean transmission rates for the sympatric and allopatric pairings were 25% and 12%, respectively, but the mean virus titers in thrips from the allopatric pairings were significantly higher than in the thrips from the sympatric pairings. This finding contrasts with our analysis comparing all transmitting and non-transmitting thrips, which showed that mean virus titers were higher in thrips that transmitted than in those that did not, and suggests that local adaptations between virus and vector leading to more efficient virus transmission involves more than simply higher virus replication or accumulation rates in the vector. In the collection locations for T. tabaci and TSWV isolates used in our study the most abundant vectors are F. fusca and F. occidentalis. The evidence for local adaptation between TSWV isolates and T. tabaci populations used in our study suggests that selection on the virus population for adaptation to specific vectors is sufficiently strong that even minor vector species such as T. tabaci can influence local vector-virus evolution21  and frequency of transmission16, virus titers in the thrips are not necessarily indicative of titers in the salivary gland and salivary reservoir from which the virus is carried in saliva to the plant during feeding. There are numerous opportunities for virus replication and movement within the vector that may be compromised, resulting in reduced transmission efficiency26. Viral genes that encode nucleocapsid, glycoproteins, and NSs have been implicated in infection and movement in thrips vectors27–31. For TSWV to be transmitted by thrips, it must traverse the midgut following ingestion by first instars and infect the muscles surrounding the midgut where replication occurs. The virus must then move via the tubular salivary glands and the efferent duct that leads from the principal salivary glands to the salivary reservoir, and the level of salivary gland infection has been shown to be a critical determinant of transmission34. Viral receptor proteins putatively interact with the receptors of midgut cells in thrips, enabling cell-to-cell movement of TSWV via endocytosis to the salivary glands for transmission29,31,35–37. Even if replication occurs, infection may be limited to the midgut if virus cannot cross the basal lamina38, and midgut-limited infections can persist in adult thrips that cannot transmit the virus, as well as in non-vector species that have fed on virus infected plant tissue40. In aphids, the basal lamina of the accessory salivary gland acts as a selective barrier associated with differential transmission of Barley yellow dwarf virus43 and specific cells in the primary salivary gland control differential transmission of Tomato yellow leaf curl China virus and Tomato yellow leaf curl virus by different whiteflies in the Bemisia tabaci cryptic species complex44.A genetic basis for differences in transmission efficiency of tospoviruses by F. occidentalis and F. fusca47 that limits virus movement, suggesting variation in vector competence may be affected by differences in immune responses of the thrips vectors. Similarly, the silencing suppressor (NSs) might also have a role, as it has also been shown to influence virus accumulation in F. occidentalis; although its exact role is unknown, it likely suppresses the immune response30.TSWV has been shown to elicit an immune response in 48, which are believed to be largely responsible for transmission because they leave cells intact49. Both infections in thrips and host plants significantly alter survival and development times of F. occidentalis and F. fusca, and the magnitude of these effects is influenced by host plant, virus isolate and temperature4. Effects of plant and vector infections on fitness of T. tabaci have not been reported, but may contribute to the lower transmission rates and higher titers observed in allopatric T. tabaci-TSWV isolate pairings. Although not formally studied, poor survival of T. tabaci on TSWV-infected plants was observed in some of the transmission experiments . Plant-virus interactions leading to changes in plant attractiveness to vectors are documented for plant viruses and their vectors50 but have not been studied in this pathosystem.Behavior and fitness-related effects were not examined in this study but have been documented to influence transmission of plant viruses by their vectors. Infection of TSWV in male thrips increased feeding and the number of non-ingestion probesT. tabaci isolines likely depends on genotypic interactions between virus isolates and T. tabaci isolines21. Our results extend these findings by demonstrating that variation in virus titer in adult thrips is not entirely responsible for the observed variation in transmission efficiency, and provide additional support for the importance of specific vector-virus interactions and co-evolutionary dynamics determining transmission efficiency within a single vector species. A better understanding of virus replication, movement, and accumulation in the vector is needed to determine whether transmission outcomes are influenced by more efficient localization or replication in the salivary glands. Understanding epidemiologically important patterns of variation in vector competence also requires a better understanding of the mechanisms underlying transmission, similarities of these mechanisms across sympatric vector species, and the influence of vector-imposed selection pressure on viral populations.Previous work demonstrated that transmission efficiency of TSWV by T. tabaci individuals and TSWV isolates were subsamples of those tested by Jacobson and Kennedy17 in their characterization of differences in transmission efficiency among different pairings of T. tabaci isolines and TSWV isolates obtained from multiple locations and host plants in North Carolina. The clonal isolines of T. tabaci were established from thelytokous females  collected at each location to minimize genetic variation within each of the isolines. Transmission efficiency was then characterized by each of the isolate by isoline pairings. Details of collection, establishment of clonal thrips isolines, TSWV isolate establishment, transmission experiments, and results from transmission assays to characterize each TSWV isolate-isoline pairing are described in Jacobson and Kennedy17. Individual thrips were classified as transmitting or non-transmitting based on a DAS-ELISA test of the leaf discs on which viruliferous thrips were allowed to feed during the inoculation access period. Samples of transmitting thrips, non-transmitting thrips, and infected leaf tissue used for acquisition of TSWV from each isolate-isoline pairing were flash frozen in liquid nitrogen and stored at −80 °C until used in the experiments reported here. Twelve of the 89 isolate-isoline pairings for which transmission efficiencies were reported by Jacobson and Kennedy17 were included in the experiments reported here; they were representative of the range in transmission efficiencies observed among the 89 isolate-isoline pairings and included both sympatric and allopatric isolate-isoline pairings17  following the protocol used by Mason et al.51 with some modifications. For homogenization, each individual thrips was placed in a 1.5 ml microfuge tube, flash frozen in liquid N2, and then homogenized with a motorized micropestle  followed by the addition of TRIzol. All incubation steps were at room temperature and all centrifugation steps were done at 16,000 x g. The pellets were air dried for 15 minutes, instead of vacuum concentrated, and resuspended in 8 µl  treated water .Total RNA was extracted from 105 individual Emilia sonchifolia source leaf tissue (20 mg) with TRIzol using the manufacturers’ protocol. Homogenization was done using three Pyrex solid glass beads  in a 1.5 ml tube containing leaf tissue, flash frozen in liquid N2, and shaken for 20 seconds in a Silamat S6 mixer . Total RNA was resuspended in 50 µl of dH2O.Total RNA was extracted from E. sonchifolia and T. tabaci. Following total RNA extraction sample quality was confirmed by running the RNA on a gel to inspect quality as well as a NanoDrop 1000  to confirm the quantity. Once the RNA extraction method was confirmed, it was used on the actual experiment samples and confirmed on the NanoDrop 1000.To ensure the integrity of flash frozen samples, nucleic acid concentrations of flash-frozen tissue were compared to fresh tissue samples for both 2O were combined to a final volume of 12 µl, heated at 70 °C for 5 minutes and chilled immediately on ice before the remaining reagents were added , Murine RNase inhibitor  and ProtoScript II reverse transcriptase . The prep was then incubated at 25 °C for 5 minutes followed by incubation at 42 °C for 1 hour. The enzyme was inactivated at 80 °C for 5 minutes and stored at −20 °C until used for RT-qPCR.cDNA synthesis was required to convert total RNA to the DNA template required for RT-qPCR. First strand cDNA was synthesized from total RNA using ProtoScript II reverse transcriptase . Thrips and leaf disc cDNA was synthesized using 2 µl of total RNA in a 20 µl reaction primed with 2 µl of random hexamers . The primers, 1 µl dNTP mix (10 mM), total RNA and dHT. tabaci. Previously reported F. occidentalis actin primers as well as other internal control genes were assayed as possible internal control genes for T. tabaci53   were also utilized to design additional primer pairs   as another possible measurement of TSWV transcript levels in both thrips and infected source leaf tissue  gene of the small (S) RNA segment57. Primer efficiency values (E) were calculated by 10(−1/slope) where the slope of the standard curve was obtained by plotting the concentration of five, five-fold dilutions of a single cDNA reaction made from thrips with a high TSWV titer against their Ct values. TSWV transcript levels were normalized to the internal controls by the inverse ratio of Pfaffl57: Einternal controlCt  /ELCt (L).Viral titer was quantified using the primer efficiency method, in which the target gene expression is quantified relative to the expression of an internal control while accounting for primer efficiencyRT-qPCR was performed on a QuantStudio 6 Flex system  with a 96-well fast block. iTaq Universal SYBR Green Supermix, SsoAdvanced SYBR Green Supermix, and SsoFast EvaGreen Supermix  were compared for their ability to detect amplification of DNA. SsoAdvanced SYBR Green Supermix was chosen because it had the most reproducible results. All samples were run in triplicate for each primer pair to control for pipetting errors, along with no-template controls Table . ReactioVirus titers were expressed as the normalized values for the relative TSWV transcript levels in the leaf tissue used as the virus source for acquisition by the thrips or in individual thrips. Titers in the leaf discs used as virus sources were log transformed (base 10) and subjected to one-way ANOVA to test for differences among isolate-isoline pairings.Further analyses examining the relationships between virus titer in individual thrips, virus isolate, and thrips isoline were conducted using the GLIMMIX procedure of the SAS system version 9.4 . Normalized values for the relative TSWV transcript levels were analyzed using a generalized linear mixed model with an assumed lognormal response distribution. An initial analysis tested a model in which the independent variables were virus isolate, isoline and their interaction. This relationship was examined further in a second model in which the interaction term was replaced with the variable “collection location,” in which virus isolate and isoline pairings were grouped as sympatric or allopatric. Similar analyses were conducted to test the association between virus titer in thrips and probability of transmission. In these analyses, transmission was treated as a binary variable with individual thrips in our subsamples classified as transmitting or non-transmitting. We could not directly test for associations between titers and transmission efficiencies of specific isolates-isoline pairings because the numbers of transmitting thrips were too low for some pairings to ensure inclusion of a sufficient number of transmitting thrips in a random sample of available thrips. Therefore, the thrips samples from each isolate-isoline pairing subjected to RT-qPCR were chosen to ensure adequate representation of transmitting and non-transmitting thrips.Supplementary Information.


Phaseolus vulgaris L.) is a major legume crop, serving as a main source of dietary protein and calories and generating income for many Tanzanians. It is produced in nearly all agro-ecological zones of Tanzania. However, the average yields are low (<1000 kg/ha), which is attributed to many factors including virus diseases. The most important viruses of common bean in Tanzania are Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) but other viruses have also been reported. There has never been a review of common bean virus diseases in the country, and the lack of collated information makes their management difficult. Therefore, this review focuses on (1) occurrence of different viruses of common bean in Tanzania, (2) molecular characterization of these viruses, (3) detection tools for common bean viruses in Tanzania and (4) available options for managing virus diseases in the country. Literature and nucleotide sequence database searches revealed that common bean diseases are inadequately studied and that their causal viruses have not been adequately characterized at the molecular level in Tanzania. Increased awareness on common bean virus diseases in Tanzania is expected to result into informed development of strategies for management of the same and thus increased production, which in turn has implication on nutrition and income.Common bean ( Phaseolus vulgaris L.) is a diploid (2n = 2x = 22) self-pollinating species that can also out-cross, albeit at very low rates , anthracnose (caused by Colletotrichum lindemuthianum), root rot (caused by Pythium spp. and Fusarium spp.) and common bacterial blight (caused by Xanthomonas axonopodis pv. phaseoli). Other important diseases of common bean in Tanzania are bean common mosaic disease , Ascochyta blight (caused by Phoma spp.), halo blight (caused by Pseudomonas savastanoi pv. phaseolicola) and leaf rust (caused by Uromyces appendiculatus) , Cowpea mild mottle virus , Cowpea aphid-borne mosaic virus , Phaselous vulgaris endornavirus 1  and Phaseolus vulgaris endornavirus 2 , Southern bean mosaic virus , Cucumber mosaic virus , Bean yellow mosaic virus  and Squash yellow mild mottle virus . Some of these viruses have been detected in Tanzania and the isolates sequenced as reviewed subsequently.Worldwide, both pathogenic and non-pathogenic viruses infect common bean crops. They include BCMV, BCMNV, Potyvirus in the family Potyviridae. The genomic RNA of BCMV and BCMNV translate into a single polyprotein that autocatalytically cleaves into 10 mature proteins: The first protein (P1), helper component proteinase (HC-Pro), third protein (P3), first 6-kDa protein (6K1), cytoplasmic inclusion (CI), second 6-kDa protein (6K2), genome linked viral protein (VPg), nuclear inclusion a (NIa), Nuclear inclusion b (NIb) and coat protein (CP). An additional short open reading frame known as Pretty Interesting Potyviridae ORF (PIPO) has been described in the P3 cistron  and Washington State University, an isolate of the serotype A virus from common bean seed produced in Tanzania was characterized and shown to cause systemic vascular necrosis (black root) at a temperature range of 23 to 27°C  , were recently discovered in common bean  is a single-stranded DNA virus with two components (A and B) and is transmitted by whitefly (Bemisia tabaci Gennadius). It has been reported to infect and cause symptoms on common bean plants in the New World  extracted from dry leaf samples  and Ugandan cassava brown streak virus (Ipomovirus), and hence studies on evolution of these viruses  computers are sufficient for most virus genome assembly work. Moreover, installing a Linux virtual machine in 1 TB HDD or less capacity computers is sufficient for analysis of NGS data using some computer programs such as VirusDetect program . This requires the shipping of RNA samples on dry ice or manipulating of RNA, which may compromise results. Unfortunately, all low-cost international couriers operating in Tanzania do not accept packing using dry ice and specialist couriers that accept nucleic acids on dry ice charge over USD3000, an amount sufficient to cover vehicle hire costs for disease surveys covering a distance greater than 3000 km.Thirdly, comprehensive countrywide surveys would require collection of many samples and their sequencing is likely to cost a huge amount of money (but some argue that it reduces costs as explained below) despite recent decreases in sequencing costs  Using NGS reduces the time required to detect viruses in many samples and thus reduces costs associated with laboratory work . (2) Only viruses detected by NGS can be specifically targeted in the steps that follow. Thus, no money is wasted on ordering primers or different antibodies to target many suspected viruses that may not be infecting samples. (3) Timely identification of a pathogen enables rapid intervention and thus may prevent spread of disease or damage to crops, which has cost implications. Therefore, the cost challenge on use of NGS may not apply in some cases and when decisions on its use are rationally made.ELISA has been used widely in detection of common bean viruses. It has been used to detect viruses in seeds  survived different environmental conditions after being air-dried. Dry leaf samples of common bean have been used for ELISA detection of common bean viruses . Petrović et al.  and non-governmental organizations such as World Vision-Tanzania and Farm Africa. The International Center for Tropical Agriculture is supporting Tanzanian organizations and private seed entrepreneurs (e.g. Meru Agro-Tours and Consultants Co. Ltd in Arusha) to produce and market bean seeds with the aim of sustaining a bean seed delivery system. Because of these efforts, quality declared seeds (QDS) is used by farmers in three zones in Tanzania: The Lake and northern and southern highlands zones . The comI and bc-12 genes that confer complete resistance to BCMV and BCMNV. Many other breeding programmes are being implemented in Tanzania; a good example is an on-going programme that aims at developing multiple-resistance (including BCMNV and BCMV) bean varieties based on Mshindi, which is an improved variety obtained in crosses that involved the variety Kablanketi , whiteflies (CPMMV and BGMV) and beetles (SBMV). Being an annual crop, vector transmission of viruses in common bean is possible through occurrence of plants infected by seedborne viruses and viruses harboured in alternative host plants, especially leguminous weeds. Indeed, low incidence of BCMNV in Malawi and southern parts of Tanzania was attributed to low vector population densities due to the high altitudes of these areas . The inoculation of BCMV and BCMNV isolates from common bean onto other legume plants caused disease symptoms on the latter  by identifying the gaps in research on common bean virus diseases, the neglected but economically important viruses such as CPMMV, CMV and SBMV)It is evident from the information obtained from literature and stakeholder consultation concerning viruses and virus diseases of common bean in Tanzania that there is scanty information concerning distribution, occurrence, characterization and detection for nearly all viruses known to cause diseases of common beans in many other countries. Although viruses that infect common beans in other countries have been characterized at the molecular level and diagnostic tools developed, in Tanzania such information is rare and when available, for example for BCMNV (Silbernagel et al., There was also scant information on alternative hosts, incidence and distribution of common bean viruses in the country. Such information is required to develop integrated pest management strategies. For instance, such information would guide deployment of specified resistant planting materials for different agro-ecological zones because information on occurrence of viruses and their strains would determine the appropriate cultivar to distribute to farmers in each area.In addition, availability of information on genetic diversity would equip common bean breeders with the tool for decisions in their breeding programmes. Whereas several common bean viruses are known to cause indistinguishable symptoms on common bean plants, different genotypes of common beans may respond differently to viruses or strains of the same virus (Feng et al., Since molecular characterization has not been done for viruses that infect common bean in Tanzania, it was recommended that NGS be considered and used in any future surveys for these viruses. This will enable detection of both known and unknown, DNA and RNA viruses. This can be followed by the use of ELISA and development of specific and degenerate primers for detection of specific or group of viruses using the sequences generated using NGS techniques.


Having an external locus of control has been associated with a range of well-supported risk correlates of offending behavior. Further, individuals with an internal locus of control orientation are suggested to be more open to engaging in treatment and are also considered more likely to have successful treatment outcomes. In forensic settings, where individuals are subject to external controls and have little personal autonomy, it is important to consider what treatment approaches might be most successful in reorienting individuals’ locus of control. The Good Lives Model (GLM) proposes a strengths-based approach to the rehabilitation of individuals who have offended. Within the GLM, an external locus of control is suggested to be associated with a deficit in the primary good of agency. In this article, we will provide a brief overview of the literature on locus of control and its hypothesized role in offending behavior. We will discuss how an external locus of control orientation is related to personal agency and how strengths-based models, such as the GLM, may assist with reorienting locus of control in individuals who have offended through promoting personal agency. The development of locus of control orientation is described within the context of post hoc reasoning about their offending and in this sense is vulnerable to cognitive processes such as justification or attempts to distance themselves from their misdeeds, as opposed to being a true reflection of their locus of control orientation generally. Therefore, it is somewhat misaligned with the social learning theory approach originally purported by Rotter, as it risks ignoring reinforcement experiences of individuals that may impact upon their beliefs relating to control and outcome expectancies, as well as their capacity for autonomy or self-direction.Locus of control is of interest to forensic practitioners due to its relationship with well supported risk correlates for offending behavior  and factors associated with treatment success . However, within the forensic literature, locus of control has been operationalized in two, arguably distinct, ways. In some instances, locus of control has been operationalized similarly to In this paper, we will first provide an overview of the hypothesized relationship between locus of control, offending behavior, and rehabilitation. We will then discuss how locus of control and personal agency are related and how it may be helpful to target the shared properties of these constructs  in forensic treatment, as opposed to simply focusing on an individual’s ability to take responsibility for their actions. Finally, we will examine how strengths-based approaches to rehabilitation such as the Good Lives Model GLM;  may helpResearch has examined the association between locus of control orientation and offense-related variables including correlates of offending, treatment engagement and change, and recidivism. Individuals who have offended tend to report having a more externally oriented locus of control than non-offending individuals, with this finding replicated across both adult and adolescent samples and a range of behaviors, including sexual offending e.g., , drunk dAlthough individuals who have offended appear to be more likely to have a more externally oriented locus of control compared to non-offending individuals, this alone is unlikely to directly account for offending behavior. An external locus of control has been found to be associated with a range of well-supported risk factors for offending. More specifically, a more external locus of control has been associated with deficits in social competency , which in turn is supported by their perceptions of and expectations of their environment , to those that are completely voluntary . More coFinally, even if individuals do decide change would be beneficial, if they do not feel they possess agency over their life, they are unlikely to feel motivated or competent enough to change. Because individuals undergoing forensic treatment are already likely to feel their autonomy is lacking, it is even more important that development of an internal locus of control is prioritized during preparation to make changes. With this in mind, the following section will explore interventions with individuals who have committed offenses that aim to shift their locus of control perception toward a more internal orientation, with a particular focus on strengths-based approaches .Strengths-based approaches represent a relatively recent development in forensic psychological practice and arose in response to criticisms of more traditional rehabilitation models, such as the Risk-Need-Responsivity (RNR) model . The RNRA number of strengths-based interventions for offending have been described in the literature, many of which are derived from the GLM . While tThe GLM  is a strIt has been suggested that one of the aims of antisocial/criminal behavior can be to demonstrate personal agency and control when individuals perceive they lack this in other aspects of their lives , making The GLM is grounded in the ethical concepts of universal human rights and human dignity . This isavoiding specific high-risk states and situations. For example, a goal for someone who has sexually offended against children might be to avoid places where children congregate, such as schools and parks, or avoiding feeling lonely, which may increase his or her risk of using the internet to access child exploitation materials. It has been argued that this focus on avoidance goals negatively impacts upon the engagement and motivation of individuals  were more likely to display a “treated profile” post treatment than those in the Relapse Prevention program (n = 321). The determination of a “treated profile” included consideration of scores on five psychometric measures including the Nowicki–Strickland Locus of Control Scale (n = 264) completed a battery of psychometrics including the Nowicki–Strickland Locus of Control Scale , In another example, The Firesetting Intervention Program for Prisoners  scores on the psychometric measures were compared with those of a comparison group (n = 45) who resided at prisons where the FIPP treatment was not available. p = 0.06) and was maintained at follow-up for FIPP participants but not for the comparison group.In another study, rs FIPP; . A batteIn addition to treatment evaluation studies, there has also been some qualitative research exploring agency, the GLM, and the process of desisting from further offending. n = 76) with that of a traditional relapse prevention approach (n = 701). Treatment effectiveness was measured by comparing attrition rates and change on participants’ pre–post treatment scores across a battery of psychometrics including the Nowicki–Strickland Locus of Control Scale. n = 52) with those of a treatment-as-usual comparison group (n = 40). It is also important to note that there has been some research that has found little or no association between GLM-informed rehabilitation and changes in locus of control orientation. For example, Justice-involved individuals often present with an externally oriented locus of control that has been associated with a range of issues including well-known correlates of offending, lower levels of motivation and engagement with treatment, and poorer treatment outcomes . Locus of control is related to the human good of agency or autonomy and is viewed as one of the needs that all individuals prioritize. Justice-involved individuals often lack the relevant internal and external capacities to achieve personal agency. For individuals with low levels of personal agency, or an externally oriented locus of control, strengths-based interventions such as the GLM promote the development of agency using a whole-program approach (from their aims and orientation through assessment and treatment) while also supporting individuals to develop the skills to continue to live a personally fulfilling life. Early research evaluating the effectiveness of GLM-consistent programs suggests that these have the potential to positively reorient agency and locus of control within forensic environments. Given forensic settings may provide experiences that further reinforce an external locus of control, GLM-informed interventions represent a potentially promising way to overcome these barriers and to support locus of control reorientation within this environment. However, despite promising early findings, research evaluating the effectiveness of strengths-based interventions, and more specifically their ability to reorient locus of control, is still very much in its infancy. Therefore, further research is needed before any definitive conclusions can be drawn about the extent to which such approaches may promote personal agency and an internal locus of control above that of traditional risk avoidant approaches.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Parthenolecanium corni (Bouché) and of grapevine leafroll-associated virus-1 (GLRaV-1) and grapevine virus A (GVA) were monitored from 2003 to 2015 in a Riesling vine plot in the northeast of France. Virus spread was compared between two periods: 2003–2008 and 2009–2014. The percentage of infected vines increased from 54 to 78% for GLRaV-1 and from 14 to 26% for GVA. The spatial distribution of viruses and of P. corni was analysed using permutation tests and revealed an aggregative pattern. Virus distribution was not associated with the density of P. corni population on grapevines. However, GLRaV-1 and GVA spread mainly from initially infected vines. New GLRaV-1 and GVA infections were more frequent on vines near primarily infected vines, first anisotropically along the row, then between neighbouring rows. Virus spread was similar to those described in literature with grapevine mealybug species. This slow vine-to-vine progression suggests that P. corni was responsible for the virus spread, in accordance with the low mobility and low transmission capacities of its local population.Distribution patterns of the European fruit lecanium  Ampelovirus genus, are transmitted by several species of mealybugs and soft scales [Vitivirus (family Betaflexiviridae). Progressive reduction of insecticide use in vineyards, changes in farming practices, and increasing commercial exchanges have favoured the outbreak of scale insect  populations. Mealybugs (Pseudococcidae) and soft scales (Coccidae) dwelling on grapevine were for a long time considered as secondary pests, until they were shown to transmit grapevine leafroll-associated viruses (GLRaVs) in different winegrowing regions of the world ,2. At let scales . Moreovet scales  assignedPietersen  and CabaThe distribution of virus-infected vines is generally random when planting material is infected at a low incidence but tends to have an even distribution when this material contains a high rate of infected vines . OccurreSystematic removal of diseased vines combined with insecticide treatments applied after the hatching of crawlers (first-instar larvae) resulted in significant decrease of viral extension ,19. In NScale insects have several ways of dispersal . In the Parthenolecanium corni (Bouché) is a soft scale widespread in vineyards of Eurasia and North America. In the northern wine-growing regions of Europe, P. corni has one generation per year, whereas in southern regions it can develop two or three generations [P. corni is one of the most common scale insect species in vineyards. In this region, GLRaV-1 is the most prevalent virus, followed by GLRaV-3 [P. corni in disease spread. Therefore, we monitored the evolution of the spatial distribution of P. corni and the virus transmitted, GLRaV-1 and GVA, in a commercial vine plot to better assess the risk of field spread of leafroll disease by this soft scale. These observations could contribute to the management strategy against the expansion of the disease and on the choice of pest control means, e.g., roguing either diseased vines only or the entire plot, protective barriers from neighbouring contaminated vines that could require a collective involvement of winegrowers on the same hillside.The coccid erations ,34,35,36erations ,38,39,40 GLRaV-3 ,42. As GP. corni, and a few individuals of Pulvinaria vitis (L) and Heliococcus bohemicus Sulc. Out of 704 vines tested, 281 were negative; GLRaV-1 is the main virus present (389 vines), either alone or in association with GLRaV-2 (10 vines), GLRaV-3 (110 vines), and GVA (138 vines). The vine plot is surrounded by other vine plots much less infested by the same species of scale insects and infected by the same viruses [P. corni crawlers [The vine plot, under organic pest management for over 20 years, is planted with cv. Riesling and located at Nothalten . It consists of 10 rows of 68 to 76 stocks south–north orientated along the slope. The 5 first pathways between rows are around 2.10 m wide, while the 4 following ones are 2.80 m. Stocks are spaced of 1.40 m within rows, except on row 6 where the 7 last vines are closer. Vine management remained the same over the monitoring period, except that since 2007 green manure replaced natural soil cover of the inter-rows. This plot shows a high density of  viruses . The oldcrawlers ,30.Infection of each stock was checked by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Tissue extracts were obtained from pooled fragments of leaves from several twigs sampled from July to October, or from dormant canes. Leaf or cane fragments (1 g leaves or 0.5 g wood for 5 mL buffer) were ground inside extraction bags with a bullet blender . Polyclonal antibodies raised against GLRaV-1, -2, or -3 or GVA were produced in the laboratory and used in a biotine-streptavidine procedure . AbsorbaP. corni was counted on each stock on the whole plant. From 2003 to 2005, spring counts were taken from studies done by Kuntzmann [Every spring from 2006 to 2015, between the last week of April and the first week of June, the number of young females of untzmann ,47,48—pountzmann  based onDistribution of viruses was mapped for each stock over 2 periods: 2003–2008 and 2009–2014. New infected vines around an initially infected vine (I) were split into 6 categories: first (I + 1) and second (I + 2) next vines in the row, and opposite  vines located on the two immediate neighbouring rows . Other p2 test.On the basis of previous studies ,10,12,50P. corni females and viruses were clustered in aggregates or randomly distributed within the plot. The spatial dependence between virus-infected vines was also tested for the 2 monitoring periods. As the local population of P. corni did not transmit GLRaV-3 in our experiments, this virus did not extend its distribution; therefore, we tested distribution of the whole infected vines over the survey period (2003 to 2014).For each mapping year, we performed permutation tests for spatial independence to assess whether young Spatial analyses were conducted using procedures designed by Peyrard et al.  and operWe computed the probability to find a vine infected by a given virus between 2009 and 2014 as a function of distance from a vine contaminated between the years 2003–2008.P. corni female numbers for 2009 to 2013.To detect the effect of a delay between virus inoculation and symptom development, we calculated the probability of virus presence on each vine during the period of 2009–2014 as a function of yearly 2), and wind speed (km/h) were obtained from a meteorological station (La Crosse Technology WS 3600) located in the vineyard of Kintzheim, a village located at about 12 km south of the experimental plot .Daily records of temperatures (°C), total precipitation  seemed to never have been colonised by this species.t border . MappingP. corni infestation  because of mortality or replacements occurring in the meantime. Out of the 682 remaining stocks, 419 (62%) were initially infected by either one or a combination of the two viruses GLRaV-1 and -3. GVA was always present in mixed infection with GLRaV-1 and/or -3, except for one vine. Only 10 vines were positive for GLRaV-2, alone or in combination with one to three of the aforementioned viruses. Distributions of GLRaV-1 and of GVA are plotted for the vines that survived over the periods 2003–2008 to 2009–2014 a,b. DistMapping of GLRaV-1 spread showed that within-row vines immediately next to an infected vine (‘first’ vines) were the most infected, then opposite vines, second neighbours within-row, and first and second vines near the opposite vines . Only ei2 = 84.1, df = 4, p < 0.001; chi2 = 29.7, df = 4, p < 0.001).Numbers of new GLRaV-1- and GVA-infected vines around initial ones were significantly different from the numbers of neighbours distributed among the five possible positions around an infected vine  or GVA (mean ± SD = 31 ± 29) in 2009–2014 was not significantly different from mean number of females on other vines .The relation between presence in vines of GLRaV-1 and GVA in the period 2009–2014 and  numbers . When thP. corni females, as well as of GLRaV-1, GLRaV-3, and GVA viruses, were monitored from 2003 to 2015 in a commercial Riesling plot at Nothalten, northeast France. The plot was heavily infested by a P. corni population that grew from 2003 to 2007, and afterwards declined progressively up to 2015. The crop was conducted under organic pest management and no insecticide was used for at least 20 years. Parasitism [P. corni density changes. Conditions of relatively high temperature and humidity are beneficial to scale insect population growth [Parthenolecanium persicae (Fabricius) showed that even at −15 °C, soft scales were still able to survive in dry conditions but were killed in wet conditions when temperatures reached −10 °C [P. corni populations, even though the winter extreme in some years reached −10 °C  [Planococcus ficus (Signoret) [Evolution of spatial distributions of rasitism  and predrasitism ,54 couldrasitism ,47,48,55n growth ,56,57, wn growth  and extrn growth . Cold tod −10 °C . At Nothd −10 °C . In the ceed 50% . Thus, eelopment ,62. Cult (Risso)  and the ignoret) . Conversignoret) ,66. In tP. corni. The results of the present study agree with aggregated spatial patterns that have been reported for other mealybug species on grapevine [P. corni, which was present at least once on almost all the stocks during our monitoring and whose distribution was aggregated. Moreover, no case of infection has been obtained after infectivity tests made in the laboratory with P. vitis and H. bohemicus nymphs sampled from the same plot [The mapping of infested vines distribution, associated with a statistical spatial analysis of females, enabled us to assess the aggregated pattern of rapevine ,67. Statrapevine ,51,68,69ame plot .P. corni numbers in years from 2009 to 2013 did not show any trend of increase with P. corni numbers. When the maximal population density was reached in 2007, infection was independent of female numbers per vine, which was not different between future infected vines and healthy vines in 2009–2014. Similarly, Charles et al. [P. corni population and virus spread explains difficulties sometimes encountered when searching for the origin of contaminations, for example, when insecticides were applied against scale insects in the meantime. When a plot becomes almost entirely contaminated, disease distribution becomes regular, making it difficult to find initial foci or a link with vector distribution.Vine infection did not appear to depend on abundance of their natural vector. Presence in vines of GLRaV-1 and GVA in the period 2009–2014 and s et al.  found noPhenacoccus aceris (Signoret) [Planoccocus spp. [P. corni showed that transmission rates ranged from 28 (with 100 L1/recipient vine) to 41% (with 50–100 L2) for GLRaV-1, and from 26 (with 100 L1) to 36% (with 50–100 L2) for GVA . Moreover, P. corni nymphs are less mobile than mealybug nymphs, and adult females are sessile. This could explain the low transmission success of this soft scale species in the vineyard in comparison with the above-mentioned species. In Italy, virus spread due to H. bohemicus was also slow [H. bohemicus nymphs showed high virus detection rates (88 to 100%), virus transmission occurred at lower rates (26%). Zorloni et al. [H. bohemicus transmitted GLRaV-3 to 2 out of 77 inoculated test plants and GVA to 1 out of 38, whereas GLRaV-1 was not transmitted.The percentage of GLRaV-1- and GVA-infected vines grew by 24 and 12%, respectively, over a 12-year period. The expansion of the viruses was slow compared to that caused by mealybug species such as ignoret)  or Planocus spp. ,13,69, flso slow , and miglso slow  observedi et al.  reportedP. corni in a new planted vine adjacent to the present study plot [P. corni populations [In our study, a few newly infected vines were recorded at over 4 m from previously infected vines. Nymphs could have been brought there by the wind, as observed with mealybugs ,28 and wudy plot ,30. The P. corni population leads us to expect that, in an already infested and infected vine plot, removal of only infected vines could be sufficient to limit virus dispersal. P. corni represents a potential threat in infected vineyards due to dispersal of its crawlers by the wind. Planting new vineyards with virus-free material (rootstocks and cuttings) is necessary to prevent the expansion of GLRaV-1 and GVA, as P. corni is widespread in the vineyard and potentially in the surrounding environment. Planting non-host green hedges [On the basis of the present study, the low rate of virus spread despite a high density of n hedges  and loca


The development of combination therapy has received great attention in recent years because of its potential to achieve higher therapeutic efficacy than that achieved by mono-drug therapy. Carriers for effective and stimuli-responsive co-delivery of multiple agents, however, are highly deficient at the moment. To address this need, this study reports the generation of multi-component hydrogel beads incorporated with reduced graphene oxide (rGO). The beads are prepared by incorporating doxorubicin (DOX)-loaded gelatine (GL) microbeads into hydrogel beads containing rGO and 5-fluorouracil (5-FU). rGO-containing beads are shown to be more effective in inhibiting the growth of MCF-7 cells via the induction of reactive oxygen species (ROS) generation. In addition, the drug release sustainability of the beads is affected by the pH of the release medium, with the release rate increasing in neutral pH but decreasing in the acidic environment. Our beads warrant further development as carriers for pH-responsive and controlled co-delivery of multiple agents. Recent years have witnessed an extraordinary expansion in the field of targeted drug delivery using polymeric matrices ,2,3. OneDespite the promise brought by advances in drug delivery as mentioned above, the efficiency of cancer therapy based on the use of chemical drugs is still unsatisfactory due to not only the low efficiency of cellular uptake and non-specific distribution in the body but also the occurrence of multiple drug resistance (MDR) . An effe2SO4), potassium permanganate (KMNO4), 30% (v/v) hydrogen peroxide, hydrazine sulphate and calcium chloride were purchased from SdFine Chemicals . DOX was received from AspiroPharma Pvt. Ltd. . Millipore water was used throughout the study.GL, graphite powder (<20 µm) and 5-FU were obtained from Sigma–Aldrich . SA, sodium nitrate, sulphuric acid  hydrogen peroxide solution. The reaction mixture was stirred for 120 min. The product was washed with distilled water to remove acid residues, centrifuged and finally dried at 80 °C for 24 h in a hot air oven. A total of 100 mg of the product was dispersed in 100 mL of distilled water and sonicated for 1 h to get a homogeneous dispersion. Hydrazine sulphate was added to the dispersion, followed by stirring for 12 h at 60 °C to get rGO. The obtained product was washed with distilled water and dried at 80 °C for 24 h in a hot air oven.rGO was fabricated by using Hummer’s method as previously described . Brieflyw/v) aqueous GL solution, followed by stirring at 300 rpm until a homogeneous solution was obtained. The solution was added dropwise into 100 mL of paraffin liquid light containing 1% (v/v) Tween 80 under constant stirring at 600 rpm. After additional 20 min of stirring, 2 mL of glutaraldehyde (GA) was added. The GL-DOX beads formed were collected, filtered and treated with n-hexane to remove oil residues. They were then dried at 40 °C in a hot air oven and placed in an airtight container until further use. The same protocol was adopted to generate GL-rGO-DOX beads but during the preparation process, rGO was added to the 8% (w/v) aqueous GL solution along with DOX.A total of 40 mg of DOX was added to 10 mL of an 8% (w/v) aqueous SA solution, followed by stirring at 300 rpm until a homogeneous solution was obtained. The solution was added dropwise into 100 mL of a 5% (w/v) CaCl2 solution. The resulting solution was kept in ambient conditions for 1 h for gelation to occur. The beads generated were collected, filtered and rinsed with distilled water. They were then air-dried and placed in an airtight container until further use. The same protocol was adopted to generateSA-rGO-5-FU beads but during the preparation process, rGO was added to the 2% (w/v) aqueous SA solution along with 5-FU.A total of 50 mg of 5-FU was added to 10 mL of a 2% (w/v) aqueous SA solution containing 5-FU and rGO. The resulting solution was stirred at 300 rpm for 30 min. After that, it was added dropwise to 100 mL of a 5% (w/v) CaCl2 solution. The resulting solution was kept in ambient conditions for 1 h for gelation to occur. The beads generated were collected, filtered and rinsed with distilled water. They were then air-dried and placed in an airtight container until further use.GL-rGO-DOX microbeads were prepared as described above and were added to a 2%  and thermogravimetric analysis (TGA) were performed by heating the sample at a heating rate of 10 °C/min under a nitrogen atmosphere from 35 to 600 °C. X-ray diffraction (XRD) analysis was performed by using an X-ray diffractometer  at a scanning rate of 10°/min using Cu Kα radiation. The morphological features of the samples were studied by scanning electron microscopy (SEM) using a scanning electron microscope .The structure of the samples was characterized by using a Fourier transform infrared (FTIR) spectrophotometer  in the wavenumber range of 400–4000 cmfm was the mass of the drug in the filtrate and dm was the total mass of the drug added during the preparation of the drug-loaded beads.A total of 10 mg of drug-loaded beads was dispersed in 10 mL of phosphate buffer saline (PBS) containing 0.5 mL of ethanol. The dispersion was stirred in darkness in ambient conditions for 24 h. After that, the beads were retrieved by centrifugation, crushed in PBS, sonicated for 10 min and centrifuged again. The supernatant was filtered. The drug concentration in the filtrate was determined by using a UV-Vis spectrometer at the wavelength of 478 nm for DOX and 270 nm for 5-FU. The EE was calculated by using the following formula:A total of 30 mg of drug-loaded beads was put into a dialysis bag, which was then immersed in 300 mL of PBS at 37 °C at various pH values . At regular time intervals, the amount of drug released from the beads was analysed by using a UV-Vis spectrometer at the wavelength of 478 nm for DOX and 270 nm for 5-FU. The release data of the drug-loaded beads were analysed by fitting them into different kinetic models  in order to evaluate the release mechanism of the drug from the beads.2 atmosphere. After that, the sample was added to each well at a desired concentration, followed by incubation of the plate at 37 °C in a 5% CO2 atmosphere for 24 h. The MTT reagent was added to each well at a concentration of 0.5 mg/mL. After incubation at 37 °C in a 5% CO2 atmosphere for 3 h, the medium in each well was removed. A total of 100 μL of DMSO was added to each well. The plate was agitated on a gyratory shaker before the absorbance at 570 nm was measured by using a spectrophotometer. The IC50 value was determined by using the linear regression equation , with the value of y being set as 50 and the values of m and c being derived from the viability graph.MCF-7-human breast cancer cells were purchased from the American Type Culture Collection  and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 6% foetal calf serum (FCS) and antibiotics as previously described . During The haemolytic activity of the beads was determined as previously described . All pro5 cells per well and were incubated for 24 h at 37 °C in a 5% CO2 atmosphere. After that, the sample was added to each well at a desired concentration, followed by incubation of the plate at 37 °C in a 5% CO2 atmosphere for 24 h. Cells were washed with PBS thrice. A total of 250 μL of trypsin-EDTA was added to each well, followed by incubation at 37 °C for 3–4 min. The cells were harvested by centrifugation at 300× g at 25 °C for 5 min. They were then treated with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (10 μM) at 37 °C for 30 min. The cells were centrifuged at 150× g for 5 min and washed with Dulbecco’s PBS (DPBS) to remove the extracellular dye. They were re-suspended in 400 μL of pre-warmed DPBS. The dichlorofluorescein (DCF) fluorescence of the cells was analysed by using a flow cytometer  at an excitation wavelength of 488 nm and an emission wavelength of 535 nm.MCF-7 cells were seeded in a 6-well plate at a density of 3 × 10–1 , 1635 cm–1 (C=O stretching vibrations of amide-I), 1458 cm–1 (C=O stretching vibrations of amide-II) and 1380 cm–1 (C–N stretching vibrations). After being loaded with DOX, the peak assigned to the stretching vibrations of C=O was shifted to 1627cm–1 due to the formation of hydrogen bonding interactions between the hydroxyl group of DOX and the NH2 group of GL. A new peak was also observed at 1735 cm–1 due to the presence of DOX in the matrix of the GL beads. These results confirmed that DOX was successfully loaded into GL microbeads.DOX-GL microbeads were synthesized and embedded into an SA solution containing 5-FU and rGO for the formation of multi-component hydrogel beads. To examine possible chemical interactions among rGO, 5-FU and DOX in the beads, FTIR analysis was performed. In the FTIR spectrum of DOX, a broad peak was observed at 3327 cm–1 A. This pbrations . On the –1 due to N–H and C–H stretching vibrations whereas the peaks at 1662 cm–1 and 1248 cm–1 were contributed by C=O stretching vibrations and C–F stretching vibrations, respectively (–1 (O–H stretching vibrations), 1601 cm–1 (C=O stretching vibrations) and 1388 cm–1 (C–O stretching vibrations) (–1 due to the occurrence of electrostatic interactions between 5-FU (–NH group) and SA (–OH group). New peaks were also noted at 711cm–1 and 1149 cm–1 due to stretching and bending vibrations of C–F. These findings confirmed that 5-FU was successfully loaded into the polymer matrix.The FTIR spectrum of 5–FU showed peaks at 2831–3138 cmectively B. The FTrations) C. After –1 and 1728 cm–1. These peaks were attributed to stretching vibrations of O–H and C=O groups, respectively  assigned to C=O stretching vibrations in the spectrum of SA beads was shifted to 1596 cm–1 in the spectra of both SA-rGO-5-FU and SA-5-FU-rGO-GL-DOX due to the interactions between rGO and the SA matrix and those between rGO (C=O group) and 5-FU (–NH group). This observation was in good agreement with that reported by Piao and Chen [–1 in the spectra of both SA-rGO-5-FU and SA-5-FU-rGO-GL-DOX due to π-π stacking interactions between rGO and DOX/5-FU. This statement was consistent with the observation made by Ma and co-workers [The FTIR spectrum of rGO showed characteristic peaks at 3317 cmectively D. The peand Chen  who note-workers  who repoApart from using FTIR, the structure of the beads was characterized by using XRD . The XRDThe morphological features of the beads were studied using SEM . All of The thermal stability of the beads and their constituents was analysed by using TGA and DSC . The TGA+ ions in the release medium disrupted the hydrogen bonding interactions between rGO and the drug, leading to a higher percentage of the loaded drug being released; whereas at pH 6.8, the formation of hydrogen bonding interactions between rGO and the drug was not disrupted and this reduced the amount of the drug successfully released from the beads [The EE of rGO-containing beads (SA-rGO-5-FU and SA-5-FU-rGO-GL-DOX) was much higher than that of the beads in which rGO was absent . This wahe beads . At pH 1he beads ,42. Such2) values, the release profiles of all drug-loaded beads  fitted the Higuchi model, suggesting that the drug release process involved the penetration of the release medium into the hydrogel matrix for the loaded drug molecules to diffuse through the pores into the external environment. The release data were fitted into the Korsmeyer–Peppas equation:tM is the mass percentage of the loaded drug released at time t, αM is the total amount of the drug loaded into the beads, k is the release rate constant and n is the diffusion exponent. The diffusion exponent values were in the range of 0.402–0.665. This suggested that the release process was mediated by the anomalous or non-Fickian type diffusion.The kinetics of drug release was determined by fitting the release data into various kinetic models  . Based oHaemolysis is an important factor determining the biocompatibility of a drug carrier. The treatment of erythrocytes with GL-DOX and SA-5-FU-GL-DOX led to a low percentage of haemolysis . Althoug2DCFDA. The cell treatment was found to increase the endogenous ROS level in MCF-7 cells  examined, the percentage of drug release was the lowest at pH 1.2. Such release behaviour is highly favourable for oral drug administration because it enables the loaded drug to be protected from the acidic gastric environment and allows the drug molecules to be released when the carrier reaches the intestinal region. Along with their high biocompatibility, as demonstrated by the haemolysis assay, our multi-component beads are promising carriers showing high potential for exploitation for future use in co-delivery of multiple agents.


The COVID-19 pandemic has shown that understanding the genomics of a virus, diagnostics and breaking virus transmission is essential in managing viral pandemics. The same lessons can apply for plant viruses. There are plant viruses that have severely disrupted crop production in multiple countries, as recently seen with maize lethal necrosis disease in eastern and southern Africa. High-throughput sequencing (HTS) is needed to detect new viral threats. Equally important is building local capacity to develop the tools required for rapid diagnosis of plant viruses. Most plant viruses are insect-vectored, hence, biological insights on virus transmission are vital in modelling disease spread. Research in Africa in these three areas is in its infancy and disjointed. Despite intense interest, uptake of HTS by African researchers is hampered by infrastructural gaps. The use of whole-genome information to develop field-deployable diagnostics on the continent is virtually inexistent. There is fledgling research into plant-virus-vector interactions to inform modelling of viral transmission. The gains so far have been modest but encouraging, and therefore must be consolidated. For this, I propose the creation of a new Research Centre for Africa. This bold investment is needed to secure the future of Africa’s crops from insect-vectored viral diseases. The ability to detect viral pathogens accurately and speedily has been thrust into global importance with the advent of the coronavirus SARS-CoV-2, the causative agent of COVID-19 disease ,4. UnforManihot esculenta) [Similar challenges can affect agriculture. Pathogens and pests can leave most parts of the globe vulnerable ,10. A seculenta) . LessonsPhaseolus vulgaris) [Zea mays) [Ipomoea batatas) [Carica papaya) [Cucurbita pepo) [Telfairia occidentalis) [Dioscorea spp.) [Vigna unguiculate) [In Africa, HTS has been used for the detection of viruses from crops such as common bean (ulgaris) ,14,15,16ea mays) ,18,19, sbatatas) ,21,22, p papaya) , pumpkinta pepo) ,25, flutentalis) , cassavaentalis) ,28,29, yea spp.) ,31, cowpiculate)  and in Piculate) . These ficulate) . MinION Commensurate to HTS, bioinformatics training for plant sciences is important for African-based researchers. Most bioinformatics training initiatives including short courses and virtual learning platforms have targeted human health interventions ,36,37. SMost laboratory plant virus diagnostics in Africa have been reliant on serology and notably Enzyme-Linked Immunosorbent Assay (ELISA) . ELISA eMost immunostrips are imported from the United States and Europe. There is almost no capacity to produce these strip tests for plant viruses anywhere in Africa, although their manufacture is not complex and some can literally be ‘’ink-jet’’ printed . The COVOryza sativa), and valuable commercial crops, such as tomato (Solanum lycopersicum) [Virus genome information has been used by Africa’s researchers to create laboratory-based molecular diagnostics utilizing polymerase chain reaction (PCR) and reverse-transcription-PCR (RT-PCR) for staples, such as cassava, sweet potato and rice (ersicum) ,49,50,51icipe) in Nairobi, have historically had the best facilities to support virus molecular diagnostics work. This is changing, notably through two initiatives in West Africa. First is the Laboratoire Mixte International PathoBios, LMI PathosBios (www.pathobios.com), in Burkina Faso. Created in 2013, as a partnership to universities of Burkina Faso with support from France and Belgium, LMI PathoBios, runs two research platforms (with molecular laboratories) on two sites in Burkina Faso. These research platforms make it possible to carry out molecular diagnostics, microbiology, and plant experimentation for researchers in Burkina Faso and other countries in the subregion. Another is the West Africa Virus Epidemiology, WAVE (https://wave-center.org/), headquartered in Cote d’Ivoire with membership from the nations of Benin, Burkina Faso, Democratic Republic of Congo, Ghana, Nigeria and Togo. To improve early detection and diagnosis of plant viruses, laboratories have been built or refurbished and equipped in all institutions hosting the WAVE program in all the member countries. In Côte d’Ivoire, diagnostics facilities comprising state-of-the-art laboratories for molecular virology were opened in 2016. These timely and impactful capacity investments are increasing the capacity of these west African nations to detect and diagnose plant viruses.However, PCR-based techniques, cannot be rapidly scaled in the event of an epidemic without exacting high cost and infrastructural demands. International organizations, such as ILRI, the Nigeria-headquartered International Institute for Tropical Agriculture and the International Centre of Insect Ecology and Physiology (www.connectedvirus.net). Through this initiative, pump-priming grants supporting 20 research projects, in 14 countries, involving 11 different crops, and collaborations of 55 researchers in 34 institutions are in various stages of execution. Through CONNECTED, training, mentorship and networking opportunities have grown. The end of its funding cycle should not lead to the demise of the gains made. Increased use of such networks increases visibility of African researchers, enhances knowledge sharing and promotes “south–south” research connections.In-depth studies on how insects vector plant viruses are a rarity in Africa. Some of these, led by the United Kingdom (UK)-based researchers have enabled a deeper understanding of virus vectoring by aphids and whiteflies and detailed mathematical modelling ,53,54. MThe key question from this synthesis is: how can we consolidate and enhance the gains in HTS, improve diagnostics and sustain studies into insect vectoring of plant viruses for the benefit of Africa’s crops? There are currently no research centres designed to host these three research areas as their core functions anywhere on the African continent. Bold proposals and investments are needed to consolidate these three research areas into long-term action so that crops protection in Africa can be proactive rather than reactive. In subsequent sections, I will use two essential staples, i.e., maize and common bean as case studies to demonstrate the value of HTS, diagnostics and studies into virus vectoring by insects.Tombusviridae) and members of the Potyviridae family: Sugarcane mosaic virus (SCMV), Maize dwarf mosaic virus (MDMV), Johnsongrass mosaic virus (JGMV) or the Tritimovirus, Wheat streak mosaic virus [In Sub-Saharan Africa, maize is a crucial staple to more than 300 million people. It accounts for 73% of the total starch demand in Eastern and Southern Africa and 64% in Western and Central Africa . Recentlic virus .The discovery of MLN in Kenya in 2011 and its rapid spread to Rwanda, Democratic Republic of Congo, Tanzania, Uganda, South Sudan and Ethiopia was a reminder of how acute food crises can be caused by viruses ,57. In KFrom early on, HTS was used to decipher the diversity of the circulating viral components of MLN. From the genome information, RT-PCR diagnostics were developed . Whole-gIn the MLN management strategy, portable diagnostic strips tests for MCMV were useful in collecting data on the spread of the virus across different countries. In addition, significant resources were used to identify maize cultivars that were resistant or tolerant to the viral components of MLN . MCMV diFrankliniella), while the potyviral components are vectored by a variety of aphids such as Rhopalosiphum maidis, R. padi, Myzus persicae and Schizaphis graminum [However, in the absence of studies into the vectoring of MLN viruses by insects, the current state of MLN in the Eastern Africa region could end up being akin to dodging a bullet. MCMV is vectored by chrysomelid beetles and thrips , a Sobemovirus, that causes up to 50% yield loss in some bean varieties ,39,66. TAphis fabae and Myzus persicae) on common bean [It is now widely accepted that viruses can affect insect choice of host plants and feeding behaviour to influence their onward transmission . Advancemon bean . They remon bean . Mathemamon bean . The insBoth cases studies reveal research gaps; vector studies, in the case of maize, and diagnostics  in common bean. Investing in a New Research Centre for Africa, anchored around three research pillars, i.e., HTS, diagnostics and plant-virus-vector interactions, is key to guiding and hosting long-term research effort to find new ways to protect Africa’s crops. The activities within the three pillars should complement each other to provide a comprehensive suite of solutions for crop protection.This research pillar aims to provide information that will ultimately forewarn and forearm farmers, policymakers and researchers on new viruses and changes in the genomics of known viruses. Advanced molecular laboratories with HTS technologies and capability are required for virus discovery through metagenomic approaches. Appropriately planned and targeted HTS activities will lead to (1) the discovery of novel viruses and detection of known viruses that could be spreading undetected, (2) mapping the species diversity of the discovered viruses and their presence in different hosts and trophic levels within cropping ecosystems and (3) providing whole-genome sequence information of the detected viruses to inform genomics-driven creation of diagnostic tools.This research pillar will enable and increase the use of diagnostics as a primary tool of viral disease surveillance and management. Rapid diagnostics will empower farmers to test for diseases on the farm as soon as they identify “disease-like” symptoms. This way, virus incidence data can be crowdsourced. Essential activities in this research pillar should include (1) development, and manufacture, of rapid diagnostic tests and kits, (2) maintenance of a growing repository of positive controls for viral pathogens accessible to researchers from all over Africa, (3) generation of PCR primers, probes, conventional PCR, RT-PCR and quantitative PCR tests and (4) research into other novel diagnostic capabilities utilizing nanotechnology.Arabidopsis thaliana) and Tobacco (Nicotiana sp.)) for experimentation, (2) maintenance of insect stocks, (3) experimental simulation of different biotic and abiotic conditions on location and (4) mathematical modelling. These capabilities, collectively, can be used to understand the threats posed by different insects with respect to virus transmission and crucially whether the interventions we devise will make things better or worse.The goal of this research pillar is to generate biological information that can be used in mathematical modelling of virus transmission by insects. This information is essential in moving farming toward sustainable production systems by preventing or reducing insect vectoring of viruses. Continual experimentation in ways of disrupting vectoring that utilize chemical ecology, barrier plants, insect host ranges and biocontrol measures is required. Facilities such as laboratories, insectaries, climate-controlled plant growth chambers, glasshouses, large insect arenas and greenhouses are needed. These facilities will enable (1) year-round growth of critical food crops and model plants , a first centre could strategically be in Kenya, East Africa. Kenya enjoys good infrastructure, a stable economic and political climate and excellent communication connectivity for IT and travel. In addition, the presence of two CGIAR centres has made Kenya a hub for international research. The new centre would be used to perfect a phased approach in assembling a core of researchers and acquisitions of equipment and technologies needed to build momentum in all three research areas. The goal would be to get from concept to a working model Centre with ongoing research along with the three thematic areas of virus discovery, diagnostics and plant-virus-vector interactions within 5 years. This “working model” can then be replicated in other African countries such that a network of fully-fledged research campuses with space, infrastructure, researchers, and resources become a reality.


Erigeron annua (L.), Canadian horseweed, Erigeron canadensis (L.) and Canadian goldenrod, Solidago canadensis (L.). These species were predominantly hosts of the aphids Brachycaudus helichrysi and Aulacorthum solani under both management systems. The 13% higher coverage of E. annua under LIF conditions resulted in a 30% higher B. helichrysi abundance and ∼85% higher A. solani abundance compared with HIF conditions. To reveal the incidence of virus infection in crop plants and invasive weeds, high-throughput sequencing of small RNAs was performed. Bioinformatics analysis combined with independent validation methods revealed the presence of six viruses, but with strikingly different patterns under LIF and HIF conditions. Their presence without symptoms in invasive weeds and crop plants supports the necessity of employing new approaches to those currently employed in invasive weed management. These findings also suggest that invasive weeds could serve as hosts for local aphid species and reservoirs for plant pathogenic viruses, both under low and high input management systems. In this light, as here demonstrated, viruses transmitted by local aphid species were found to differ between the management systems; hence, the importance of B. helichrysi and A. solani as virus vectors in particular clearly needs to be re-evaluated. Altogether, we accept that the present study is a pilot one and individual virus vectoring of aphids still needs to be directly tested. Even so, it represents one of the first contributions to this particular area, and thereby paves the way for further similar applied research in the future.In the present study, we conducted field surveys to detect the population density of the most important invasive weed species and their associated virus vectoring aphids in crops grown under high input field (HIF) vs. low-input field (LIF) conditions, with and without fertilizers and pesticides. The most frequent invasive weed species were annual fleabane,  Invasive weeds represent a serious threat to world crop production as global trade expands and climatic conditions shift . In thisErigeron annua (L.), Canadian horseweed, Erigeron canadensis (L.), and goldenrod, Solidago canadensis L., occur in all types of habitat and are considered to be the most important and economically-relevant invasive weeds within the agro-ecosystem  to assess the population density of the most important invasive weeds under differently managed agricultural systems  vs. no chemical management ; (2) to identify and compare population densities of the most frequent aphid species on the most frequent invasive weeds; and (3) survey and identify plant viruses using high-throughput small RNA sequencing of invasive weeds and surrounding crops under different management regimes.Field surveys were conducted over a 2-year period during the crop growing seasons of 2015 and the 2016 in Central and Eastern Transylvania, Romania. The methods used have also been described by 2 at an altitude ranging from 230 to 800 m above sea level (a.s.l.) and characterized by a landscape mosaic of different land-cover types . The farming practices are predominantly small scale subsistence farming, with no chemical inputs. One consequence of this kind of land use is the exceptional biodiversity and natural value of the farming landscape  of the Old Saxon cultural region covering an area of ∼ 7,440 kmandscape . Howeverandscape .2 at altitudes between 220 and 750 m a.s.l. has been, and continues to be, intensively treated with synthetic fertilizers and pesticides, major crops including maize, potato, and alfalfa  annua (L.), Canadian horseweed, Erigeron (=Conyza) canadensis (L.), and Canadian goldenrod, Solidago canadensis (L.). These species are known to grow in a diverse range of habitats and are considered important weeds in Europe, causing substantial crop losses following colonisation of new areas  and the similarly polyphagous foxglove aphid, Aulacorthum solani   . These aZea mays . VirusesZea mays . Host pl legumes . The mos legumes , 2014.Firstly, we selected two blocks of land approximately 3 km long and 1 km wide inside each management system (regions); these blocks were located 10 km distant from each other in LIF and in 15 km distant in HIF. Inside each block, we further established two transects (at least 1 km apart) of 10 m long × 1 m wide at an approximately equal distance (between 1 and 2 metre) to three major crops  dominating (as crop) more than 95% of both management systems at field crop margins. In this way, each transect was surrounded by at least 8–10 ha of high-input, and at least 0.5–3 ha of low-input agricultural crops, including around weed populations dominated by maize, alfalfa and potato fields. The transect selections were made at the same period each year (between 5 and 8 May) and when all crop plants from both regions were at the same vegetation stage. The same transect and the same crop plant combinations were followed the next year. Each transect was carefully measured and located using GPS.2 quadrats were placed. Each of these was further subdivided into 10 × 10 cm sub-quadrats (100 sub-quadrats inside each quadrat). Inside each of these sub-quadrats all plants (native and invasive) were counted and their coverage estimated  and one species which had a coverage between 15 and 20%. Therefore, from each sub-quadrat, out of the 10 plants sampled, at least eight belonged to the dominant species and one or two to the second most dominant species.Because plants contained aphid colonies, and the exact number of individual aphids was important, all plastic bags were labelled and kept at low temperature (∼0–4°C in a cool box), then returned to the laboratory, whereupon all samples were stored at −20°C, with aphids later counted and species identified . In totaAssessment began at the end of May and was repeated fortnightly five times during the summer until the end of the weed growing season, whereupon no more aphids were found. The whole procedure was repeated in the following year using the same collection methods within the same transects. All aphids were carefully counted under laboratory conditions, and the plant materials were used for virus identification employing metagenomic high-throughput sequencing (HTS) of small RNAs as an unbiased method, able to detect all viruses present in the sample .Leaf samples on which aphids had been found and counted were used for viral analyses from both weeds and crop plants  and both management systems, with small RNA analyses performed in 2017. RNA was extracted using a phenol-chloroform method . BrieflyFor small RNA HTS, small RNA was isolated from polyacrylamide gels involving RNA pools which were prepared by mixing equal amounts of RNA originating from different individuals, collection times (in the case of weeds) and from different species in the case of crops . This poGSE132755.These pools were used for small RNA library preparation  using Truseq Small RNA Library Preparation Kit  and our modified protocol . SamplesPooled RNA extracts or RNA extracts prepared from the individual crops were used as templates for cDNA synthesis by a RevertAid First Strand cDNA Synthesis Kit  with random primers according to the manufacturer’s instructions. The generated cDNA was then used for PCRs  at 65°C, washed according to the manufacturer’s instructions and exposed to X-ray film.For Northern blot analysis, 4 μg of total RNA  were separated on a formaldehyde-containing 1.2% agarose gel and blotted to a Hybond-N membrane. Radioactively labelled random DNA probes were generated from cloned, purified PVX PCR product with a Decalabel DNA Labeling Kit (Thermo Scientific). Northern blots were hybridized with this probe in Church buffer , 1 mM EDTA, 0.25 M Na2 sub-quadrat was determined by averaging the plant values from each 10 × 10 cm plot. Next, the inter-annual differences in coverage were tested using multivariate analysis of variance (MANOVA) and mean coverage values obtained for one 1 m2 quadrat (40 data/field type/collection dates) were considered. No significant difference in weed coverage were detected between years (P = 0.12). Therefore, data from the 2 years, collected on the same dates, were combined for the analyses. The weed frequency data were tested using Poisson-distributed errors residuals for normality of errors (Kolmogorov-Smirnov test) and for equality of variance (Levene’s test). Because residuals did not meet the assumption of normality, we used the non-parametric Kruskal-Wallis- and Mann-Whitney U test to compare variables. Weed species and management systems (HIF vs. LIF) were used as fixed factors and the average weed coverage in 1 m2 sub-transect as random factor.For weed data, the mean coverage per 1 mB. helichrysi and A. solani. General linear modelling was used with mean aphid abundance on E. annua, E. canadensis and S. canadensis as response variable. Initial analyses indicated no difference between study years and aphid abundance averaged across study years (P < 0.23). The model included cropping system type (HIF vs. LIF), aphid species (B. helichrysi and A. solani), and their interaction as explanatory variables. Because aphid abundance is a discrete variable, Poisson-distributed errors were assessed. Aphid abundances on E. annua was normally distributed, so factorial ANOVA was used, followed by Tukey testing. Aphid abundance on E. canadensis and S. canadensis did not meet the assumption of normality, hence the Kruskal-Wallis test was used, followed by the Mann-Whitney U test.We next determined how the cropping system differentially affected associational susceptibility to the two aphid species, P < 0.05) interactive effects (cropping system type × species) suggested that the effect of cropping system depended on aphid species. Aphid density analyses on crop plants were made considering only the abundance of B. helichrysi. As the data was normally distributed, ANOVA was used, followed by Tukey testing to compare abundances between treatments and crop plants. The density of A. solani was only high in potato, but there were no significant differences between treatments (T-test P = 0.78); therefore no other analyses at this species abundance on crops were made. All analyses were performed using R version 3.0.1 (Macrosiphum spp.) were detected, and we did not include them in the analyses.Significant  was used to identify the proportion of variation in each PCA axis (Aphids density and treatments) explained by each virus distribution. We then used the average count of each virus reads numbers detected and log10 transformed from each weed and crop plant sample grouping as response variables, and used aphid abundance as component 1 (PCA axis1) and treatment (LIF vs. HIF) as component 2 (PCA axis 2) scores for each virus reads as independent variables. RNA and DNA viruses were analysed separately, and the only one insect virus detected  was not considered in our analyses. PCA covariance analyses were run using Community Analysis Package 4 (Pisces Conservation Ltd).de novo assembly) and compared the resultant contigs using BLAST to the NCBI Reference Genomes of plant viruses downloaded from GenBank. In parallel, we directly mapped contigs to Reference Genomes of those viruses which were represented at least with one contig in any of the libraries mapping to the reference tool of CLC Genomic workbench. Virus presence was recorded if at least two parameters were reached, i.e. virus specific contig was present and/or normalized redundant virus specific read count was >200, and/or coverage of the virus genome was >60%.Virus diagnosis was determined by small RNA HTS. For bioinformatics analysis of the HTS results, we used CLC Genomic Workbench. Briefly: for trimming, quality control and QC reports, embedded protocols in CLC Genomic Workbench were employed. For virus diagnostics, we followed two strategies and used CLC Genomic Workbench: we built longer contigs from the non-redundant reads using assembler of CLC  and HIF treatment regimes (84.5%). Two other species were present at lower densities. S. canadensis was only present in LIF, with a coverage of 2.5%. No other invasive weeds were assessed under this management system. E. canadensis was only present under HIF with a coverage of 15%. Other weed species, mostly amaranth, Amaranthus spp. in HIF regimes with an average coverage of 0.5%, were observed at the end of the growing period of the above mentioned weed species. Dominance of E. annua was significant under both management systems     . No other differences between treatments were observed in alfalfa  and potato   . The nex< 0.001) . A highe < 0.01) . Further < 0.01) . A very  < 0.91) .de novo 3,553–19,038 longer contigs  coincided with a relatively high (>200) normalized redundant read and >>60% coverage of the genome.Annotation can lead to false positive results in familiar viruses . MoreoveHelicoverpa zea (Lepidoptera: Noctuidae) feeding in the fields sampled. Of plant viruses, PVY and TVCV were the most widespread, only lacking from library 6_SS and 2_TB, respectively. Crops grown under LIF contained nine viruses, while under HIF, only six were found. PVX and BLRV were present only in crops under HIF, while PVM and PBCoV were only present in LIF crops , whilst treatments (LIF vs. HIF) had only a smaller effect (26%) . In contE. annua in LIF further resulted in a significantly higher B. helichrysi abundance (about 30% more). The same trend was observed for A. solani, where a 13% higher coverage of E. annua resulted in an increase of about 85% for this aphid under LIF compared to HIF regimes  regimes. As determined experimentally, a 13% higher coverage difference of  regimes . In our  regimes , it was detected . TherefoB. helichrysi, can be explained in all three crop plants  and the high habitat diversity apparently may influence associational susceptibility of crop plants, and the presence of the two aphid species, especially p plants .per se, and DNA virus distribution  between weeds and crops by contrast predominantly influenced by the management system involved  is probably more influenced by aphid density involved . While winvolved  clearly Besides aphid-transmitted viruses, another important group of plant pathogenic viruses comprises those transmitted mechanically or indeed, by unknown means. In the case of PVX virus, as here presently found only in HIF regimes, if the tubers are infected, cultivators can readily disseminate it within and between potato crops. With PVM, it is usually present in herbaceous Solanaceous plants within the surrounding flora. We didn’t detect it in any of the Composite weeds sampled and tested, probably because they cannot host it. However, it was only present in the LIF regimes, suggesting that it could be transmitted by the sap sucking herbivorous insects like aphids feeding on these weeds. The same could happen with BLRV and PBCoV: since the Composite weeds do not host them, we hence failed to detect them in the libraries.DNA viruses were detected in the crops within the LIF regimes, and contrastingly were present in weeds in both HIF and LIF, but here revealed a different pattern of infection . This reLastly, several of the viruses detected in our study have not previously been reported from Europe, i.e. Opuntia virus X, Tobacco vein clearing virus, Sweet potato symptomless mastrevirus 1, Pineapple bacilliform comosus virus, Sugarcane bacilliform Guadeloupe D virus and the Helicoverpa zea nudivirus 2. Although our detection of these viruses by small RNA HTS seems credible, further studies involving a different approach (e.g. RT-PCR) need to be performed in order to add support to these findings. What is certain is that the presence of these viruses in both invasive weeds and crop plants without the production of pathological symptoms in the former raises the clear necessity of acknowledging the potential and indeed likely presence of such pathogenic disease reservoirs during invasive weed management scenarios.A. solani, which is highly polyphagous. Because of the unpredictable high local aphid density on invasive weeds, aphid migration onto several local crops  may potentially cause — and indeed probably does often cause — unpredictable virus infections, including under low-input management regimes. From this we deduce that cultivation methods involving invasive weed and insect vector control need to be reconsidered, even when and if no other management approach is planned. To this end, further research is planned to quantitatively test viral infestation and detect virus spread via weed-aphid-crop plants.Overall, we conclude from these findings that even if low-input management farming systems are widely studied e.g.  and are Whilst this pilot present study is not definitive in that individual virus vectoring aphids were not directly tested, nevertheless it represents one of the first contributions to this particular topic area. As such, the study details the pioneering nature of this broad approach, one that shows considerable promise in attempts to understand the role of weed reservoirs in weed-crop aphid borne virus transmission, thereby paving the way for the next publications planned from our group in this fascinating area of applied research.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The manuscript does not contain any studies with human participants performed by any of the authors.A-KS, AB, and JK perceived and designed the experiment. ÉV, AH, and ED performed the small RNA HTS. A-KS, JB, A-KS, and AB performed the data collections. ZG and ED performed the RT-PCR. AH performed the Northern blot analysis. A-KS, AB, and HL performed the data analyses, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.