The selection of the adequate viruses is important since they must infect and preferentially replicate in both the host plant and the insect vector. The routes of transmission that determine the extent of the infection inside the insect vary among different plant viruses. In the context of the proposed strategy, plant viruses that are capable of transversing the insect gut-hemocoel barrier and replicating in insect tissues are attractive candidates. Thus, the transmission of such viruses in a persistent and propagative manner is considered as a prerequisite for this strategy to be feasible, a characteristic that is found in viruses from the families Bunyaviridae, Reoviridae, and Rhabdoviridae. In addition, several RNA viruses are known that replicate in both plant and insect tissues via a yet unclarified transmission route. In this review, advances in knowledge of trans-kingdom transmission of plant viruses and future perspectives for their engineering as silencing vectors are thoroughly discussed.Plants, plant viruses, and their vectors are co-evolving actors that co-exist and interact in nature. Insects are the most important vectors of plant viruses, serving as both carriers and hosts for the virus. This  Studies on plant viruses’ biology have shown their dependence on a plethora of vector organisms for their transmission to a new host. This vector repertoire involves insects, mites, nematodes, plasmodiophorids, and fungi . AlthougHowever, different strategies  are emplTrans-kingdom virus-induced gene silencing” (TK-VIGS) , two main strategies can be followed regarding the introduction of the “silencing” fragment into the viral genome, as described for plant closteroviruses : (1) An TK-VIGS) , as outlPlant viruses that are known to be transmitted via particular insect families have been observed to follow diverse routes in their vectors’ bodies . This diBemisia tabaci and the Lettuce infectious yellow virus (LIYV) , the virus is acquired during the probing and feeding activity. In the case of the non-persistent way of transmission, plant virus acquisition by stylet piercing of plant tissue is followed by transmission to the next plant by piercing within a period of seconds to minutes. This non-persistent strategy is mainly used when aphids repetitively puncture cells during the probing of plant tissue en route to feeding with phloem sap . For plas (LIYV) . In thatHowever, the most interesting category for biotechnological applications is that of plant viruses that are transmitted in a persistent manner, with considerable variation in terms of transmission window. This transmission route is also characterized as circulative, because the acquired virus must transverse the gut epithelium from the alimentary canal to other tissues and organs, so that it can finally reach the salivary glands. Depending on the particular plant virus–insect interaction, the transmission process may rely on specific viral proteins. Extended duration of feeding on infected plant phloem (hours), as well as prolonged retention time and a latent period, is required in order to obtain adequate transmission efficiency of the virus. Circulative plant viruses that do not replicate in the insect vectors include luteovirids, geminiviruses, and nanoviruses and are transmitted by aphids, whiteflies, and leafhoppers . The capReoviridae, Bunyaviridae, and Rhabdoviridae families, as well as the Bunyaviridae-related genus Tenuivirus , such as those belonging to nuivirus . In addinuivirus . Plant vBy definition, plants lack any ability to move and they depend on their root system that provides connection with the soil in order for a plant to feed, grow, and remain alive. So, for any virus that inhabits a plant, it is normal to rely on mobile vectors so that it is transmitted to other plant hosts.Tospovirus genus (Bunyaviridae)  and Thysanoptera (6), which are characterized by pierce-sucking mouthparts . It mustviridae) .More vectors have been observed in other orders too  , in whicAn important feature that defines the suitability of plant viral vectors for the delivery of the dsRNA silencing signal to their target insect regards the ability of the virus to replicate or not in the insect cells. Most plant viruses are not able to replicate in their insect vectors, so they can only be considered as vehicles for transfer of dsRNA that has already been synthesized inside the plant to the insect. On the other hand, replicating viruses not only are able to transverse the gut-hemocoel barrier (as “persistent circulative” viruses mentioned earlier) but also adopt a propagative strategy, which means that dsRNA intermediates are produced during their life cycle.Bunyaviridae , Reoviridae , and Rhabdoviridae . Also Tenuivirus genus  belong to this type of viruses ssDNA genome, such as Tomato yellow leaf curl virus (TYLCV) transmitted by different whitefly species], Luteoviridae , and Nanoviridae .On the other hand, viruses that circulate in the insect body but in a non-propagative, thus not-replicative, manner includes members of the families of Phytoreovirus genus and tymoviruses are transmitted by leafhoppers (Cicadellidae); and reoviruses of the genera Fijivirus and Oryzavirus as well as tenuiviruses are transmitted by planthoppers (Delphacidae). A notable exception, however, is the rhabdoviruses that can be transmitted either by aphids, leafhoppers and planthoppers RNA strands are encapsidated in a subviral particle, where they are transcribed into (−)RNAs, so that dsRNA is then produced by base-pairing of the complementary (+) and (−) strands. In the case of single-stranded or ambisense RNA viruses, intermediate replicative steps normally may also lead to the temporary production of dsRNA molecules. Engineering of plant viruses that possess single-stranded RNA genomes with appropriately designed sequences in order to transcribe RNA molecules containing self-hybridizing regions would lead to the formation RNA hairpin structures. These RNA hairpins could then be processed in dsRNAs and siRNAs by the RNAi machinery of the insect vector. Also, regarding DNA viruses inverted repeats can be introduced in their sequence so that RNA hairpins with dsRNA structures can be formed upon transcription.Laodelphax striatellus by the Rice black-streaked dwarf virus  or RSV (Tenuivirus)    after infection with H. vitripennis reovirus (HoVRV) (Phytoreovirus genus)  in the insect vector, it can be assumed that the RNAi silencing machinery is turned on upon such an infection. Several examples of vsiRNA production in insects during plant virus infection have been reported in the literature, such as the infection of the small brown planthopper uivirus) , of the by RBSDV , in the viridae) , and in viridae) . Furthers genus)  althoughs genus) .However, while deep sequencing efforts have detected vsiRNAs of propagative plant viruses in hemipteran vectors, their functionality (capacity of gene silencing) remains largely unexplored. Thus, a systematic effort needs to be initiated regarding the role of RNAi as an antiviral defense mechanism against replicating plant viruses in insect vectors, which will include the effect of knockdown of the RNAi machinery on viral replication  and As a first straightforward application of engineered plant viruses causing gene silencing in insect vectors, the VDPS system  was deveTobacco mosaic virus  that produced RNAs in sense or antisense orientation targeting actin, chitin synthase 1, and V-ATPase genes of the hemipteran pest Planococcus citri that were initially inoculated to Nicotiana benthamiana plants through agroinfiltration. This method was successful in causing silencing of the above-mentioned genes of the insect vector  among grapevine plants , tomatillo , and tobacco (Nicotiana tabacum) plants. In the same study, two other viruses  were tested . In these studies, the Bursicon, V-ATPase and Chitin synthase 1 genes of the insect vector were targeted, as proven by decrease in expression and increase in population mortality as well as other phenotypic effects , was used to create an RNAi vector for the silverleaf whitefly Bemisia tabaci where numerous cDNA clones could be then inserted and used as a screening method for finding the ideal target gene  was effectively used to silence the altered wing disc (Awd) gene in CTV-based RNAi assays against the hemipteran phloem-sap sucking insect Diaphorina citri, although this particular virus is known to normally encode for three different silencing suppressors. Interestingly, these experiments involved a few additional steps, as CTV constructs were first agroinfiltrated into N. benthamiana plants, and virions produced there were then isolated and inoculated to Citrus macrophylla plants to trigger silencing in its target insect D. citri  technology to combat insect vectors was the construction of recombinant t vector  that is e plants . Anothere tested . A few y effects , 2018. Aget gene . Also CiD. citri .In the above examples, plant viruses were engineered as a VDPS to produce dsRNA in plant tissues at high levels to cause gene silencing and mortality in insects after feeding. The RNAi effects in the insect vectors were not caused by plant virus replication within the vectors but by the large amounts of dsRNA produced in the plant cells. VDPS is not considered a static system since it can be envisioned that the VDPS systems and the viruses that sustain them are spread through the plant populations by taking advantage of the virus transmissibility by insect vectors . HoweverTymoviridae family that are known to infect plants but are also non-persistently transmitted by phloem-sucking insects (Culex Tymoviridae-like virus (CuTLV) was isolated a few years ago from mosquitoes in China and found to present high sequence similarities with other plant-infecting Tymoviridae genera  and Bombyx mori macula-like virus (BmMLV) in honeybee and silkworm samples, with the genus Maculavirus being one of the main genera of Tymoviridae family, thus meaning a possible interplay of these virus between hosts of plant and insect origin (Flock house virus (FHV) is an insect pathogenic virus that belongs to the Nodaviridae family, but can also replicate and produce functional virions in plants  is another plant virus for which a reverse genetics system became available  vectors during their transmission and its efficiency therefore is limited to particular insect species that are competent hosts for propagative transmission. Trans-kingdom VIGS is therefore more likely to be developed as a “tailor-made” application for the control of particular plant virus–insect vector combinations.In comparison to VDPS, however, Trans-kingdom VIGS.To achieve such goals, much more knowledge needs to be acquired for the establishment of reverse genetics systems for propagative plant viruses and the stimulation of the transmission between plant hosts and insect host/vectors. Furthermore, viruses  exist that have a very broad host range and that can replicate in both plants and insects also deserve renewed attention to see if they can be adapted to the strategy of AK, DK, and LS conceived the idea, designed the study, and wrote the manuscript. DK prepared the figures of the study. All authors read and approved the final version of 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.


Identifying whether novel human viruses disproportionately originate from certain animal groups could inform risk-based allocations of research and surveillance effort. Whether such “special reservoirs” exist remains controversial. We show that the proportion of viruses that infect humans varies minimally across reservoir taxonomic orders. Instead, the number of human-infecting viruses increases proportionately to the total number of viruses maintained by each reservoir group, which is in turn explained by the number of animal species within each group. This supports a host-neutral explanation for observed variation in the number of zoonoses among animal groups, such that traits of animal orders are unlikely to produce viruses that disproportionately threaten humans. These findings refine strategies to identify high-risk viruses prior to their emergence. The notion that certain animal groups disproportionately maintain and transmit viruses to humans due to broad-scale differences in ecology, life history, and physiology currently influences global health surveillance and research in disease ecology, virology, and immunology. To directly test whether such “special reservoirs” of zoonoses exist, we used literature searches to construct the largest existing dataset of virus–reservoir relationships, consisting of the avian and mammalian reservoir hosts of 415 RNA and DNA viruses along with their histories of human infection. Reservoir host effects on the propensity of viruses to have been reported as infecting humans were rare and when present were restricted to one or two viral families. The data instead support a largely host-neutral explanation for the distribution of human-infecting viruses across the animal orders studied. After controlling for higher baseline viral richness in mammals versus birds, the observed number of zoonoses per animal order increased as a function of their species richness. Animal orders of established importance as zoonotic reservoirs including bats and rodents were unexceptional, maintaining numbers of zoonoses that closely matched expectations for mammalian groups of their size. Our findings show that variation in the frequency of zoonoses among animal orders can be explained without invoking special ecological or immunological relationships between hosts and viruses, pointing to a need to reconsider current approaches aimed at finding and predicting novel zoonoses. Filoviridae, Orthomyxoviridae, and Togaviridae contain a large proportion of zoonotic species , and otherwise excluded (n = 2), creating a final dataset of 429 reservoir–virus associations. Data on zoonotic status  were obtained from Olival et al. .We used literature searches to expand an existing dataset of the maintenance cycles of single-stranded RNA viruses  to inclul et al. . Cetartil et al. A. We qual et al. , here del et al. B. The mal et al. A and B. l et al. B. Both tl et al. C. The tal et al. A. For exWe next used generalized additive mixed models with zoonotic status as a binary response variable to identify and rank the host and/or viral traits that influenced the zoonotic status of viruses. When ranking models using the Akaike information criterion (AIC), the top model explained a total of 52.3% of deviance in zoonotic status A. This mSI Appendix, Fig. S2).Among effects related to reservoir hosts, zoonotic risk declined with greater phylogenetic distance from reservoirs to primates, but this effect explained only 0.7% of the deviance in the top model and was absent from other competitive models A and E. SI Appendix, Fig. S3). In contrast, viral family effects were robust to inclusion of general reservoir effects . When the family-specific reservoir effect was not present, viral rather than reservoir taxonomy was the stronger predictor of zoonotic status . Thus, our results provide no support for the special reservoir hypothesis, which predicted that viruses associated with certain reservoir groups would be more likely to be zoonotic.We further evaluated whether effects of reservoir hosts on zoonotic status might have been missed due to the inclusion of potentially collinear variables or insufficient statistical power to fit complex models. First, given that study effort understandably increased for zoonotic viruses, including this factor might have disguised true reservoir effects. However, models which did not adjust for virus-related publication counts failed to support reservoir effects; the best of such models achieved similar AIC values with and without reservoir effects . These models outcompeted those including previously identified effects of virus-related research effort and the phylogenetic distance of each reservoir group from primates .Instead, both the total number of viruses associated with each reservoir group  and the number of zoonoses matched expectations under the reservoir richness hypothesis. The absence of reservoir effects meant that viral richness was strongly correlated with the number of zoonotic species . The only potentially “special” reservoirs identified by this analysis were the cetartiodactyls  and primates, which hosted more zoonoses than would be predicted for mammalian groups with their species richness . Consequently, the proportion of cetartiodactyl viruses which were zoonotic was unexceptional, inconsistent with the special reservoir hypothesis , Nipah henipavirus (Paramyxoviridae), and Ebola viruses (Filoviridae)  , 19, 31.viridae) , 33, theLyssavirus, which contained 73% of the 25 zoonotic rhabdovirus species and is strongly bat-associated . The putatively heightened frequency of zoonotic rodent-associated picornaviruses might conceivably arise if transmission by rodents selects for viral traits that disproportionately promote zoonotic capability or if picornaviruses have similar zoonotic capability across reservoir groups but heightened ecological overlap favors zoonotic transmission from rodents. Yet, given that our dataset contained only three rodent-associated picornaviruses  and primates and cetartiodactyls also hosted zoonotic picornaviruses , a statistical artifact of low sample size cannot be ruled out. In contrast, effects of nonhuman primate reservoirs on the zoonotic potential of adenoviruses and retroviruses may have a biological basis. All primate-associated adenoviruses (three out of three) and 7 out of 12 primate-associated retroviruses were zoonotic, while none of the 15 adenoviruses and 16 retroviruses associated with other reservoirs have been reported to infect humans. This suggests that evolutionarily conserved similarities between nonhuman primates and humans may facilitate zoonotic transmission in these viral families. Yet, our results also highlight limitations of host phylogeny as a generalizable predictor of zoonotic capability since primate associations did not affect the zoonotic status in most of the viral families maintained by nonhuman primates. We hypothesize that the advantages conferred by phylogenetic relatedness are crucial for viral families that are inherently host-restricted  but are less influential for viruses with less specialized infection and replication mechanisms. More generally, the relatively poor performance of phylogenetic distance in predicting either the proportion or the number of zoonoses from different reservoir groups . Among rhabdoviruses, a significantly elevated proportion of bat-associated species were zoonotic, but this effect was driven by the rabies-causing genus sociated . Howeverr groups A and B sr groups , 37 are Our modeling framework allowed us to distinguish the relative contribution of host and virus features to zoonotic status. Consistent effects of viral taxonomy support the conclusion that zoonotic ability is a feature of viral clades rather than host associations , 28; howThe number of zoonoses scaled positively with the host species richness of animal orders . Since rSI Appendix, Fig. S6). Second, we considered a single reservoir order for most viruses rather than modeling every association of each virus with each infected host species within that order independently. This was critical to avoid potentially spurious effects driven by surveillance effort and multiple observations of the same virus in closely related species. For example, Dengue virus (DENV) has been detected in at least 20 bat species, which comprise 76% of DENV–host associations . One possible explanation for the apparent discrepancy between our findings and those based on shared virus detections could be that heightened surveillance for or susceptibility to zoonoses has led to elevated detections of spillover infections in previously identified “special hosts.” Our results highlight the importance of separating exposure and infection from transmission in future studies which involve reservoirs of infection.Our dataset and analytical approach differed in several epidemiologically important ways from previous studies which suggested variation in viral zoonotic propensity across animal groups , 12. Firciations , 12, butciations –43. Our A clear limitation of our approach was that we were unable to consider traits that vary within reservoir groups  which may moderate the baseline zoonotic risk by altering viral richness, transmission dynamics, or ecological contacts of hosts with humans . In addiIn summary, our analysis suggests that variation in the frequency of zoonoses among major bird and mammal reservoir groups is an emergent property of variation in host and virus species richness. We find no evidence that intrinsic or ecological differences among animal groups increases the number of viruses they maintain or the likelihood that any given virus is zoonotic. Basing public health surveillance and research strategies aiming to identify high-risk viruses on the assumption that some taxonomic orders of hosts are disproportionate sources of zoonoses risks missing important zoonotic viruses while simultaneously reenforcing patterns that may reflect detection biases rather than zoonotic risk.Adenoviridae, Anelloviridae, Arenaviridae, Arteriviridae, Asfarviridae, Astroviridae, Birnaviridae, Bornaviridae, Caliciviridae, Circoviridae, Coronaviridae, Filoviridae, Flaviviridae, Genomoviridae, Hantaviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Nairoviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Peribunyaviridae, Phenuiviridae, Picobirnaviridae, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, Tobaniviridae, and Togaviridae)  , 46. Forviridae) .Fields Virology  and otherwise discarded (n = 72). Yellow fever virus, for example, can be maintained by humans but also has an independent nonhuman primate reservoir community, and lineages from such maintenance cycles are zoonotic  and Woolhouse and Brierley (n = 70) with additional literature searching (n = 3). Only detections where the identity of the virus infecting humans was confirmed to species level by PCR or sequencing were considered.Following Babayan et al. , data onVirology , followeVirology . We suppVirology  with 344on cycle . Nineteezoonotic . Two virl et al.  (n = 342Brierley  (n = 70)We used logistic regression models to assess the association between reservoir group and zoonotic status (a binary response variable). Because at least one previously reported predictor of zoonotic status—publication count—was expected to be nonlinear , we usedThe simplest reservoir effect was a random intercept for each reservoir taxonomic order, with all reservoir orders assumed independent. This represented the typically assumed special reservoir effect, in which some reservoirs are more prone to maintain zoonotic viruses than others. A second representation allowed clustering on the reservoir phylogeny to represent the hypothesis that related reservoir groups have correlated associations with zoonotic viruses. For example, mammalian reservoirs may be associated with a larger fraction of zoonoses than avian reservoirs, or related mammalian clades may share phylogenetically conserved features which shape their association with zoonoses. This phylogenetic random effect was implemented as a multivariate normal distribution, where phylogenetic relationships among taxa determined the amount of correlation between randomly sampled intercepts for different reservoir taxonomic orders . The varhylogeny , 52. We hylogeny . Followihylogeny , this phhylogeny . The thiCercopithecine gammaherpesvirus 14 and Hare fibroma virus, neither of which have publicly available sequence data in GenBank, meaning their reported reservoir associations are questionable). To capture a measure of the virus-related research effort in each reservoir group, a set of queries of the form “<taxid>[Organism:noexp] AND virus” were constructed, replacing “<taxid>” with the NCBI Taxonomy ID of each taxonomic order in turn. For nonhuman primates, this query was modified to “(txid9443[Organism:noexp] NOT txid9606[Organism:noexp]) AND virus,” where “txid9443” is the Taxonomy ID for the order Primates, and “txid9606” is the Taxonomy ID specific to Homo sapiens. Both variables were included as the natural logarithm of the respective publication counts, reflecting our prior belief that the effect of increasing numbers of publications would become saturated at high values. Nevertheless, we also allowed for additional nonlinear effects by fitting both variables as thin-plate smooths with 10 and 8 knots for virus and reservoir publication counts, respectively  virus species and 2) reservoir group (on 1 November 2019). For each virus species, this query was “<taxid>[Organism:noexp],” where “<taxid>” was replaced by the NCBI Taxonomy ID corresponding to each virus species. Viruses with no entry in the NCBI taxonomy database were removed from all analyses  received a value of 0.8. Virus species sharing no taxonomic levels were treated as independent (similarity = 0). Missing taxonomic assignments were interpolated to retain a comparable number of levels across all viruses, ensuring that all viruses remained independent at all levels not supported by the official ICTV taxonomy. For example, since many viruses are not classified using suborders a gap between order and family was bridged by creating a new level, assigning each family to its own unique suborder. This approach generated several free-floating branches, which are assumed independent in our models, but this is consistent with current virus taxonomy .The taxonomic random effect of virus family used the same specification as the reservoir phylogenetic effect above. Because the included virus families were too diverse to be represented in a single phylogeny (and might not share a common ancestor), a variance–covariance matrix representing currently accepted virus taxonomic relationships was generated. This matrix reflects the proportion of the taxonomy that is shared between virus families. Specifically, pairs of viruses were assigned a similarity score N/max(N), where N is the number of taxonomic levels they share, and max(N) the total number of taxonomic levels. This calculation was performed considering all taxonomic levels from realm to family. Thus, viruses in the same virus family received a similarity value of 1, while viruses from different families in the same order  were fit to the counts of each virus type using the MASS library in R . Both seThe distribution of viral diversity among reservoir groups was quantified by calculating profiles of normalized alpha diversity using the rdiversity library in R . These chttps://doi.org/10.5281/zenodo.3516613.Data and code used in this manuscript are available at Supplementary File


Fusarium culmorum strain A104-1 originally sampled on wheat in Belgium. This novel virus, for which the name Fusarium culmorum virus 1 (FcV1) is suggested, is phylogenetically related to members of the previously proposed family ‘’Unirnaviridae’’. FcV1 has a monopartite dsRNA genome of 2898 bp that harbors two large non-overlapping ORFs. A typical -1 slippery motif is found at the end of ORF1, advocating that ORF2 is translated by programmed ribosomal frameshifting. While ORF2 exhibits a conserved replicase domain, ORF1 encodes for an undetermined protein. Interestingly, a hypothetically transcribed gene similar to unirnaviruses ORF1 was found in the genome of Lipomyces starkeyi, presumably resulting from a viral endogenization in this yeast. Conidial isolation and chemical treatment were unsuccessful to obtain a virus-free isogenic line of the fungal host, highlighting a high retention rate for FcV1 but hindering its biological characterization. In parallel, attempt to horizontally transfer FcV1 to another strain of F. culmorum by dual culture failed. Eventually, a screening of other strains of the same fungal species suggests the presence of FcV1 in two other strains from Europe.A new mycovirus was found in the  Fusarium has received much attention leading to the discovery of many mycoviruses, especially in Fusarium oxysporum and Fusarium graminearum [F. culmorum.Mycoviruses have been described in all major fungal taxa and more extensively in genera with economic or medical importance . Amongstminearum ,3. In thF. culmorum belongs to the fungal complex causing Fusarium head blight and Fusarium foot and root rot on several plant species [F. culmorum is known to produce mycotoxins that contaminate crop products, hampering their consumption by humans and cattle [F. culmorum has developed related resistance [ species . In addid cattle ,6. Curresistance . Thus, tsistance , motivatF. culmorum, no viral sequence has been described yet [The majority of known mycoviruses are made of dsRNA , hence sibed yet ,10. By sFusarium culmorum virus 1 (FcV1) was recovered from the F. culmorum strain A104-1. This strain was originally sampled in Corroy-le-Grand (Belgium) on wheat in 2008. Identification and handling of the fungus was done as previously described [g for 1 min, the supernatant was collected and subjected to ethanol precipitation. The obtained pellet was resuspended in a final volume of 15 µL of milliQ water.escribed . DsRNA eescribed . The dsRE. coli 10β cells. The plasmids were subsequently sequenced using the forward and reverse M13 universal primers. Then, primers corresponding to the viral sequences were designed and used to obtain the sequence of the gap between the obtained amplicons.A volume of 5 µL of the purified dsRNA solution was used to perform a reverse transcription using the M-MLV reverse transcriptase  with the degenerate primer 5′-GACGTCCAGATCGCGAATTCNNNNNN-3′, according to Wang et al. . The res2-3′ was ligated on both extremities of the dsRNA using T4 RNA ligase , O/N at 4 °C. Reverse transcription was then performed using the complementary primer 5′-GGCAATTAACCCTCACTAAAG-3′, with the same enzyme already mentioned. Subsequently, the cDNA was amplified using the primer 5′-TCACTAAAGAATTCGATCGATC-3′ in combination with the viral specific primers 5′-TCCGAAAGGATGCTCG-3′ or 5′-GAATGAGTTTGGAATGAACATT-3′. Amplicons were directly sequenced.The 5′- and 3′-termini were sequenced according to Xie et al. . Brieflyhttp://www.bioinformatics.nl/cgi-bin/emboss/plotcon, with default parameters) and the prediction of coiled coil was made using Deepcoil [The whole genome of FcV1 was assembled using UGene . The RNADeepcoil . Remote Deepcoil  with theDeepcoil  and usedDeepcoil , with ulDeepcoil . The treDeepcoil . AlternaDeepcoil  and visuF. culmorum strain A104-1 was grown on synthetic nutrient-poor agar (SNA) medium for 10 days at room temperature (RT). Macroconidia were harvested by adding and collecting 1 mL of sterile water containing Tween-20 (one drop per mL) to the cultures. The suspension was then poured on 3% (w/v) agar plates with serial dilutions and incubated O/N at RT. Sixty germinated macrononidia were individually transferred onto potato dextrose agar (PDA) plates and subsequently incubated for 10 days at RT. Total RNA was extracted from the produced mycelia using Trizol reagent  according to the manufacturer’s instructions. Then, RNA was subjected to RT-PCR using the specific primers 5′-GGCGTTAAGGCGTTGGGCTACT-3′ (positions 1701–1722) and 5′-CCTCGACCCGCAACGCC-3′ (positions 2200–2184) with the enzymes already described.The Cycloheximide and rifampicin were used in the aim to obtain a virus-free isogenic line of the fungus. Briefly, 20 mL of carboxymethyl cellulose (CMC) liquid medium supplemented with either cycloheximide (1 mg/L) or rifampicin (1 mg/L) were inoculated with plugs of mycelia and incubated at 120 rpm at 25 °C in darkness. After 5 days of culture, conidia were collected and poured onto PDA supplemented with the same chemicals. Fourteen individual fungal colonies were isolated for each treatment and sub-cultured for at least three serial passages via hyphal-tipping on similar media. Afterwards, mycelia were grown on PDA without chemical and screened for the presence of FcV1 by RT-PCR as already described.F. culmorum strain P1P2 that was obtained from the strain UK99 after repeated passages on tebuconazole supplemented media [A mycelia plug of the fungal strain A104-1 was grown on PDA next to a mycelia plug from the ed media . Seven dFifty-three strains of F. culmorum originated from diverse locations  were groF. culmorum strain A104-1, one major band of ca. 3 kb was visible on agarose gel in addition to fungal DNA and RNA (Fusarium culmorum virus 1 (FcV1) is proposed. The full sequence has been deposited in the Genbank database under the accession number MN187541. A BLASTn search for the total nucleotide sequence resulted in significant hits  corresponding to members of the proposed family “Unirnaviridae”. Like other members of this group  corresponding to the predicted ORF1p of other unirnaviruses. All those proteins are similar in size  and show 34–61% pairwise identity . Those proteins are longer  than unirnaviruses ORF1p but contain a similar CC in their respective C-terminal region  from a phlebo/tenuivirus during a genomic recombination event, involving a partitivirus as the donor for the replicase . This hyl region . They weLipomyces starkeyi strain NRRL Y-11557 [−16, 55% coverage). A total of 153 out of 187 aa (82%) of this protein shows strong similarity to the conserved C-terminal region of unirnaviruses ORF1p, although its total length is much shorter. Such a result could correspond to an endogenous virus-like element (EVE), resulting from the integration of a viral cDNA into its host genome.Unexpectedly, a hypothetical protein (ODQ71866.1) from  Y-11557  was alsoL. starkeyi genome in the scaffold 8 at positions 129932–130495, on the minus strand . However, the remaining sequence does not match to any other characterized protein. Nevertheless, it is tempting to speculate that this protein might have been involved in the horizontal gene transfer event that led to the viral endogenization.To get more insights on this potential EVE, we used the MycoCosm portal . We retr−16) at positions 239–433. Inside this RdRp domain, the palm motifs A, B and C [As mentioned above, FcV1 ORF2 is probably translated by a -1 PRF to produce the fusion protein ORF1+2p. While the function of FcV1 ORF1p in the viral cycle remains elusive, FcV1 ORF1+2p is likely to be associated with viral replication. Indeed, ORF2 sequence contains a predicted RNA-dependent RNA-polymerase (RdRp) domain , Hubei partiti-like virus 59 (HPLV14) and Leshenault partiti-like virus (LPLV) ,45. HoweTotiviridae, all the aforementioned viruses are actually more related to members of the family Partitiviridae and a group of bipartite viruses which were previously referenced as “CTTV-like viruses”, named after the famous Curvalaria thermal tolerance virus [Despite exhibiting the genome organization of ce virus . Both viF. culmorum strain A104-1 were isolated and allowed to produce new mycelium. An RT-PCR screening showed that 100 % of those progeny mycelia were infected with the virus, highlighting the ability of FcV1 to be efficiently vertically transmitted in its host.In order to assess the vertical transmission rate of FcV1, sixty macroconidia produced by the Since conidial isolation did not result in the loss of FcV1, the fungal host was treated with two different chemicals known for their ability to cure of mycovirus, namely ribavirin  and cyclAs an alternative to the production of a virus-free isogenic line of the strain A104-1, another way towards the biological characterization of FcV1 would be the infection of a virus-free strain. A dual culture combining the strain A104-1 and the selectable strain P1P2 was achieved to this end. In practical terms, both strains were cultured on PDA and mycelia plugs from the confrontation zone were subcultured on selective media. None of the produced mycelia gave positive signal for FcV1 by RT-PCR. At this point, it is unclear whether this lack of transmission is due to the inability of FcV1 to be transmitted in this fashion or if viral transfer is impeached by vegetative incompatibility between both fungal strains.F. culmorum strains was also investigated. In order to do so, fifty-three strains that were originally sampled in Europe, Russia, Turkey, New Zealand and Canada were tested. Interestingly, two European strains gave the amplicon with the expected size (499 bp) by RT-PCR and exhibited a similar ca. 3 kb band when analyzed for the presence of dsRNA  [Wickerhamomyces ciferrii. More generally, many EVE have been found in yeasts [Debaryomyces hansenii produces a counterfeit viral capsid protein derived from an EVE that interferes with the assembly of a similar virus, acting therefore as an antiviral immunity system [L. starkeyi could act in a similar way would be worth testing. But at first, confirming the existence and expression of the detected EVE by (RT)-PCR is a prerequisite. In this study, we also reported the detection of a unirnaviral EVE in the genome of the yeast  (UvNV1) . Indeed,n yeasts ,52, as wn yeasts . Sometimy system . WhetherSouthern tomato virus was shown to be seed-transmitted at rates ranging from 70 to 90% [We were unable to cure the strain A104-1 from FcV1 by isolation of macroconidia. This vertical transmission rate of 100% was already observed for UvNV1 . In para0 to 90% . AltogetSince we were not able neither to cure the virus from the strain A104-1 nor to infect another strain, the biological effect of FcV1 remains undetermined. Previously, it was shown that ChNRV1 induces conidial defects on its host strain mutated for dcl1, whereas it seems to be asymptomatic in the wild-type strain . As far F. culmorum and we gave evidences for the presence of the virus in two other strains from Europe. In the future, it will be interesting to obtain the sequences of those potential FcV1 isolates as well as to test more strains in order to shed light on the nucleotide diversity of this virus.Eventually, we performed a screening of other F. culmorum. To our knowledge, it is the first full genomic sequence for a virus infecting this fungal species. Our studies also broaden the diversity of mycovirus belonging to “Unirnaviridae” and supports the proposition of this taxon being considered as a new viral family.In summary, in this study we have described FcV1, a novel monopartite mycovirus infecting


Nephotettix cincticeps (Hemiptera: Cicadellidae), is a key insect vector transmitting rice dwarf virus (RDV) that causes rice dwarf disease. We discovered a novel iflavirus from the transcriptomes of N. cincticeps and named it as Nephotettix cincticeps positive-stranded RNA virus-1 (NcPSRV-1). The viral genome consists of 10,524 nucleotides excluding the poly(A) tail and contains one predicted open reading frame encoding a polyprotein of 3,192 amino acids, flanked by 5' and 3' untranslated regions. NcPSRV-1 has a typical iflavirus genome arrangement and is clustered with the members of the family Iflaviridae in the phylogenetic analysis. NcPSRV-1 was detected in all tested tissues and life stages of N. cincticeps and could be transmitted horizontally and vertically. Moreover, NcPSRV-1 had high prevalence in the laboratory populations and was widely spread in field populations of N. cincticeps. NcPSRV-1 could also infect the two-striped leafhopper, Nephotettix apicalis, at a 3.33% infection rate, but was absent in the zigzag leafhopper, Recilia dorsalis, and rice Oryza sativa variety TN1. The infection of RDV altered the viral load and infection rate of NcPSRV-1 in N. cincticeps, for which it seems that RDV has an antagonistic effect on NcPSRV-1 infection in the host.The green rice leafhopper,  It has long been acknowledged that insects are important vectors and hosts of numerous viruses infecting humans, animals, and plants. Many viruses are found because of the symptomatic diseases they caused . For exaIflaviridae is a family classified under the order Picornavirales, comprising viruses with monopartite, single-stranded, positive-sense, and non-segmented RNA genomes. Currently, the family Iflaviridae consists of one genus Iflavirus, with 15 classified members. To date, all the reported iflaviruses infect exclusively arthropod hosts, especially insects. The insect hosts of iflaviruses have been found in the orders including Hemiptera, Lepidoptera, Hymenoptera, Coleoptera, and Diptera , Nilaparvata lugens honeydew virus-2 (NlHV-2), Nilaparvata lugens honeydew virus-3  into functional viral proteins (IRES[L-VP2-Vp4-VP3-VP1/-Helicase(2CATPase)-protease(3CPRO)-RdRp(3DPol)]3'UTR-poly(A)  .Nephotettix cincticeps (Hemiptera: Cicadellidae), is one of the important pests on rice in Asia and could lead to the loss of rice yields . RDV, a double-stranded phytoreovirus of the family Reoviridae, is the pathogen of rice dwarf disease and mainly transmitted by N. cincticeps in a persistent-propagative manner (N. cincticeps by a complex host-virus-vector interaction (The green rice leafhopper (GRLH), e yields . N. cince manner . RDV inferaction . It is oN. cincticeps and named it as Nephotettix cincticeps positive-stranded RNA virus-1 (NcPSRV-1). NcPSRV-1 shares a typical iflavirus genome organization. The sequence alignments and phylogenic analysis suggest that NcPSRV-1 belongs to the family Iflaviridae. Our findings reveal the characteristics of the viral prevalence, transmission, and co-infection with RDV of NcPSRV-1.In the present study, we assembled and characterized the genome sequence of the picorna-like virus discovered from the transcriptomes of RDV-infected and uninfected N. cincticeps, and two laboratory populations of the zigzag leafhopper Recilia dorsalis (Hemiptera: Cicadellidae) and the two-striped leafhopper Nephotettix apicalis (Hemiptera: Cicadellidae) were used in this study. For N. cincticeps, population A was originally collected from rice fields on the Zijingang Campus of Zhejiang University, Hangzhou, China in October, 2014 and was reared on healthy rice plants (Oryza sativa variety TN1). Population B was originally collected from rice fields on zhe Zijingang Campus of Zhejiang University, Hangzhou, China in November, 2015 and was reared on healthy TN1 rice plants. Population C was originally collected together with population B at the same time and fields and was reared on RDV-infected TN1 rice plants. Population D was originally collected together with population A at the same time and fields and was reared on RDV-infected TN1 rice plants. The RDV infection rates of N. cincticeps in population D were more than 90%. NcPSRV-1 negative population was originally collected from rice fields in Deqing, Zhejiang, China, in July, 2020 and was reared on healthy TN1 rice plants after NcPSRV-1 detection.Five laboratory populations of the green rice leafhopper R. dorsalis, the population was originally collected from rice fields on the Zijingang Campus of Zhejiang University, Hangzhou, China in 2016. The population of N. apicalis was originally collected from rice fields in Xundian Hui and Li autonomous county, Yunnan Province, China in 2019.For All laboratory populations were maintained within insect-proof cages in the climate chamber  and keptN. cincticeps using TRIzol . RNA concentration of each sample was determined using a Nanodrop 2000 spectrophotometer . Reverse transcription reactions  was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit  according to the manufacturer’s instructions and the synthesized cDNA was amplified as a template for polymerase chain reaction (PCR).Total RNA was extracted from the 5th instar nymphs of Salivary glands of the 5th instar nymphs from population A and D were collected for RDV-uninfected and infected transcriptome analyses with three biological replicates, respectively . Based on the virus genome-like sequences identified in RNA-seq assembled sequences, 15 pairs of primers  were desThe 5' and 3' ends of the viral genome were confirmed by 5' and 3' rapid amplification of cDNA ends (RACE) according to the instructions of SMART RACE cDNA amplification Kit . The amplified 5' and 3' RACE products were cloned into pRACE vector and sequenced.via Vector NTI  sequence were predicted using Protein BLAST on NCBIRNA extraction and cDNA synthesis were performed as described above. Primers NcPSRV-6F/NcPSRV-6R and NcPSRV-15F/NcPSRV-15R  were useN. cincticeps samples using virus-specific primers NcPSRV-1S/NcPSRV-1A  was performed to quantify the viral loads of NcPSRV-1 in different cPSRV-1A , which wActin-1A  as the rt method .N. cincticeps, brains, salivary glands, midguts, fat bodies, and carcasses were dissected from the 5th instar nymphs, ovaries from females, and testes from male adults. The insects collected from population A were anesthetized on ice before dissection. The dissection was performed in phosphate buffered saline  using a pair of forceps under dissecting microscope . The tissues were instantly moved into TRIzol and 50 tissues of one kind were pooled as one biological replicate. Three biological replicates were performed. The viral loads of NcPSRV-1 in different tissues were quantified using qRT-PCR.To detect the spatial distribution pattern of NcPSRV-1 in N. cincticeps, eggs, the 1st-5th instar nymphs, female and male adults were collected from population A and transferred into TRIzol. The eggs were collected from different egg masses. Fifty eggs/nymphs or adults (per stage/sex) were pooled as one biological replicate and each life stage was performed in triplicate. The total RNA of different life stages was extracted. The cDNA synthesis and qRT-PCR of different samples were conducted as described above.To detect the developmental expression pattern at different stages of NcPSRV-1 in N. cincticeps field populations, N. cincticeps populations were collected from different locations in China in 2017, including Hangzhou , Jinhua , Wuhan , Hefei , Guiyang , Fuzhou , Changsha , Kaifeng , Guangzhou , and Hechi . NcPSRV-1 infection was determined at the individual levels and more than 30 individuals were tested for each field population . The infection rates of NcPSRV-1 in population A were examined again in 2018 (n = 48), 2019 (n = 52), and 2020 (n = 46), respectively. Total RNA extraction and cDNA synthesis of the 5th instar nymph individuals were performed as described above. The viral loads were quantified with qRT-PCR as described above. The prevalence of NcPSRV-1 was calculated as previously described (Detection of NcPSRV-1 in Field Populations of N. cincticeps section).All laboratory populations of N. cincticeps. The honeydew was excreted by N. cincticeps of population A on the field around the rice plants. After being exposed to the air more than 1 week, the honeydew crystals were formed, and then collected and dissolved in 100 μl RNAse-free water. The dissolved honeydew crystals were used for total RNA extraction and cDNA synthesis. NcPSRV-1 was detected using PCR as previously mentioned in Virus Detection and Quantification section.To investigate the role of honeydew in horizontal transmission route of NcPSRV-1, the presence of NcPSRV-1 was tested in the honeydew of via the virus contaminated food, the 2nd instar nymphs of N. cincticeps from NcPSRV-1 negative population were fed on virus containing artificial diet at 25°C for 24 h. The artificial diet was a mix of 10% sucrose solution and crude virus filtrate at a ratio of 10:1. The 10% sucrose solution (W/V) was filtered through 0.22 μm filter  under aseptic conditions. The crude virus filtrate was prepared using NcPSRV-1-infected 5th instar nymphs of N. cincticeps. After frozen in liquid nitrogen, 30–40 nymphs per 2.0 ml microcentrifuge tube were ground and 1 ml PBS was added into each tube. After resuspension, the homogenate was agitated at 4°C for 15 min and centrifuged at 4°C at 16,000 × g for 10 min. The supernatant was collected and subsequently filtered through 0.22 μm filter. Then the filtrate was concentrated using Ultra-15,100 kDa Centrifugal Filter tube  and diluted to the preconcentration volume by adding 10% sucrose solution. This step was repeated twice and the crude virus filtrate was prepared. The feeding apparatus was a transparent tube (8.5 cm in diameter and 15 cm high), covered with two layers of stretched Parafilm  sealing 2 ml artificial diet inside. The feeding apparatus was sterilized before use. During feeding, the apparatus was covered with black cloth except for the Parafilm area. After feeding for 24 h, the insects were transferred to fresh rice seedlings. Insects were collected 12 days postinoculation (dpi). The insects under the same conditions but without oral inoculation were used as a control. The filtrate of uninfected N. cincticeps was prepared with the same method. PCR and qRT-PCR were conducted for the virus detection as described above.To examine the horizontal transmission of NcPSRV-1 Newly emerged individuals from NcPSRV-1 negative population and positive population (population A) were used to investigate the transmission modes of NcPSRV-1. The pairs of ♀+/♂+, ♀+/♂−, ♀−/♂+ and ♀−/♂− were mated and NcPSRV-1 infection rates of the 5th instar offspring were detected using qRT-PCR and PCR.n = 70 eggs per replicate) were tested, three replicates of the non-treated eggs (n = 70 eggs per replicate) were used as controls. To investigate the viral multiplication in eggs, the eggs without red eyespot (3 days after oviposition) were collected in triplicate (n = 70 eggs per replicate) from population A to compare NcPSRV-1 loads with the eggs with red eyespot (7 days after oviposition). The eggs with and without eyespot are shown in To confirm the potential vertical transmission routes , NcPSRV-1 infections in eggs were further analyzed. The eggs with red eyespot (7 days) collected from population A were transferred into the sterile cell strainer and submerged in 100 ml of 1–2.33% sodium hypochlorite for 10 min to eliminate potential external viral contamination. The bleach solution was pipetted up and down until the eggs sank to the bottom of the strainer. Then the eggs was lifted from the bleach solution and washed with running water for 3 min followed by washing twice with 100 ml sterile deionized water. After thorough rinse, the eggs were air-dried and transferred to sterile 2.0 ml centrifuge tubes. Three replicates of sodium hypochlorite-treated eggs . On the 12th dpi, leafhoppers of the two species were collected to test for the presence of NcPSRV-1, respectively.The 2nd instar nymphs of Rice plants were also tested for the existence of NcPSRV-1. The 5th instar nymphs from NcPSRV-1 positive population A were fed on rice seedlings (age 10 days) for 48 h. On 12 dpi, the seedling surface was washed by 1–2.33% sodium hypochlorite and flushed with water. After thorough rinse, the seedlings were sampled. The non-feeding rice seedlings at the same-day age were sampled as a control. The total RNA extraction, the cDNA synthesis, and virus detection of all the samples mentioned in this section were conducted as described above and NcPSRV-1 existence was tested using both PCR and qRT-PCR.beta-actin were amplified in the qRT-PCR using the primers described above. RDV was quantified using RDV-specific primers RDV-S8-S/RDV-S8-A . The mean of the NcPSRV-1/beta-actin mRNA ratio in the virus detection of population A in 2017 was compared with that in 2018, 2019, and 2020 by using Student’s t-test , respectively. Student’s t-tests were also used to evaluate the virus abundance between two populations with or without carrying RDV, and NcPSRV-1 titers in different groups of N. cincticeps eggs. Variable homogeneity tests were performed before the Student’s t-tests. All statistical tests were performed using Data Processing System (DPS) software . For the qRT-PCR results of NcPSRV-1 distributions, data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test  and 1,043 nt in length] were initially identified from the transcriptomes of RDV-uninfected and RDV-infected cted one . Proteincted one  and RACEThe NcPSRV-1 genome (GenBank accession number MW197427) is 10,524 nt in length excluding the 3' poly(A) tail and is A/U rich  similar to other iflaviruses. The genome contains a single ORF . The ORF encodes one large polyprotein of 3,192 aa and was flanked by UTRs. The 5' UTR is 629 nt in length and contains 13 AUG triplets. The 3' UTR of NcPSRV-1 is 327 nt in length located at positions nt 10,209–10,524 and followed by a poly(A) tail.Conserved domain analysis revealed NcPSRV-1 shared a typical iflavirus gene organization . The str3FxFx7Gx5YxP, and FxRG which have been observed in picornaviruses capsid proteins VP2, VP3, and VP1, respectively (X/DXP), which has been experimentally confirmed , motif B (1623Qx5DD1631), and motif C (1667KKx4Px5NSN1681), while the motif C of NcPSRV-1 is slightly different from the consensus sequence KGx4Sx5STN 2,997] and motif VI  was found at the location prior to the motif I of RdRp domain , protease (Pro), and RdRp from N- to C-terminus. The helicase domain was identified at residues 1,551–1,721 and belongs to the helicase superfamily 3. This superfamily is associated with NTP-binding and characterized by three conserved motifs: motif A  and aa 2,290–2,307 (YKWIFVSAAAIIGGFFLAT), followed by 3C-protease proteolytic sites [AxPE/G (A/M)] AKPEectively . These tectively  and someectively , is absePicornavirales. Fifteen member species and 13 unclassified viruses in the family Iflaviridae and the type species in other families of Picornavirales were selected for analysis. NcPSRV-1 is clustered with the members of Iflavirus . The low viral loads were observed in the brain, testis, carcass, and ovary. The viral load in testis was higher than ovaries.The relative abundance of NcPSRV-1 in the brain, salivary gland, midgut, fat body, carcass, ovary, and testis of  tissues . The traN. cincticeps was determined using qRT-PCR. The viral titers were at the similar levels and exhibited no significant differences among the different developmental stages . These results indicate that NcPSRV-1 is present throughout all developmental stages of N. cincticeps at a relatively stable viral level , 2019 , and 2020  were examined for the virus infection. NcPSRV-1 could be vertically transmitted from the mated pairs ♀+/♂+ and ♀+/♂−. The transmission efficiency attained 100% among the offspring of the infected females and males, higher than 80% in the mated pairs ♀+/♂− . When thvia transovum (on the surface of eggs) or transovarial (within the eggs) way. No significant difference was observed between the sodium hypochlorite-treated eggs and the non-treated eggs  and R. dorsalis (n = 36) from labarotory populations were tested for the existence of NcPSRV-1. The results showed that no virus positive individuals were detected in the nymphs from these two species  and C (n = 42) were determined. The infection rate of RDV was 45.24% in population C, while NcPSRV-1 was present at 9.52%. In the RDV negative population B, 56.00% individuals were NcPSRV-1 positive. The viral loads in populations B and C were significantly different , Bombyx mori iflavirus (BMIV), Lymantria dispar iflavirus 1 (LdIV1), Heliconius erato iflavirus (HeIV), and Thaumetopoea pityocampa iflavirus 1 (TpIV1) discovered in Lepidoptera insects had identical conserved motifs, sharing high similarities in the capsid protein sequences, while Lepidoptera-infecting iflaviruses Ectropis obliqua picorna-like virus (EoPV), Perina nuda virus (PnV), IFV, Opsiphanes invirae iflavirus 1 (OiIV-1), Spodoptera exigua iflavirus 1 (SeIV-1), and Spodoptera exigua iflavirus 2 (SeIV-2) were similar to each other but presented low degrees of sequence conservation compared to the viruses mentioned above, even though BMIV and IFV infected the same host Bombyx mori. The variation in VP1 may be due to the differences lying in the pathogenicity, virulence, and host range. Whether there is a direct linkage between the pathogenicity and the sequence variation in VP1 needs further verification.In previous studies, the iflavirus capsid proteins were compared with the conserved motifs observed in the family aviruses . PairwisN. cincticeps were relatively stable, exhibiting the adaptability to the insect host. Like NcPSRV-1, many iflaviruses appeared to be distributed in multiple tissues and could be detected in multiple life stages of the hosts, however, the spatial distribution tendency of iflaviruses varied. The crippled bees were strongly DWV-positive in all body parts, while the asymptomatic bees were never virus positive in the head , the first reported bee virus, made the honeybee larvae sac-like and failed to pupate and eventually died at the last larval stage  RDV may compete with NcPSRV-1 for the resources used for viral replication in the cytoplasm of the infected cells; (ii) the immune response triggered by RDV in N. cincticeps may affect the proliferation of NcPSRV-1; (iii) the alteration in nutrition or immune signaling pathway caused by continuous infection of RDV in rice may influence the insect host, therefore, impacting on the replication of NcPSRV-1. Whether there was a direct or indirect interaction between NcPSRV-1 and RDV require further investigation. Iflaviruses are often found co-infected with other pathogens. The honey bee, A. mellifera, and ectoparasitic mite, V. destructor, are commonly infected by multiple viruses, including IAPV, BQCV, DWV, and SBV associated with colony collapse disorder  of SeMNPV  and RDV (double-stranded RNA virus) in the fields or laboratory populations, respectively ,C. Especder CCD; . Severalder CCD; . Cases oder CCD; . When inf SeMNPV . The repf SeMNPV . And alspro and intermediate 3CDpro (Taken together the characteristics of NcPSRV-1, extensive geographical distribution, sustainable increasing infection rates in population of high density, covert and persistent infection, horizontal and vertical transmissions, and systematic infection, we think NcPSRV-1 or iflavirues of the similar traits has the great potential to be engineered. For instance, the iflavirues may be modified to deliver genes of interest. The positive-sense single-strand RNA virus genome, which serves as mRNA for the IRES-mediated translation of polyprotein after release, could be reformed as a vector carrying target genes. The target genes could be replicated and translated simultaneously with the engineered viral genome during the viral replication cycle and released into the cytoplasm by cleavage of the 3Ce 3CDpro  with theBased on the iflaviruses reported previously, we speculate iflaviruses exist universally in insects and many of them are mutual symbiotic or in harmony with their hosts without causing obviously damage to host fitness. The insect iflaviruses may be invertebrate-specific and widespread in the field populations. These traits may indicate that the iflaviruses infecting asymptomatically may be a part of the host virome. Furthermore, insect iflaviruses co-infect with diverse pathogens, and several cases of iflavirus-pathogen interactions have been reported. Iflaviruses could even affect the behavior of the hosts or the parasitoid wasps of the hosts. Insects, especially those in Hemiptera and Diptera, are the natural vectors of numerous pathogenic viruses and of great importance to agriculture and public health. All these properties make insect iflaviruses a reservoir for manipulating insect vectors or the route of pathogen transmission. The further study of iflaviruses will provide us new perspective and open up new possibilities for the integrated pest management in the future.Iflavirus. NcPSRV-1 was abundant in salivary glands and propagated stably in all life stages of N. cincticeps. NcPSRV-1 was wide spread in the fields populations and could maintain a relatively high infection rate when the host N. cincticeps was reared at high density. NcPSRV-1 persisted in a covert infection in the host and could be transmitted both horizontally and vertically. Co-infection with RDV was discovered and RDV seemed to have an antagonistic effect on the NcPSRV-1 infection.A novel positive-sense single-stranded RNA virus named NcPSRV-1 was identified and characterized in this study. The virus genome is 10,524 nt in length encoding one large polyprotein and shares a typical iflavirus genome arrangement. Multiple sequence alignment and phylogenetic analysis showed that NcPSRV-1 has high sequence similarity with Hubei picorna-like virus 29 and clusters with the members of the genus The datasets presented in this study can be found in the GenBank, NCBI. Accession numbers are listed in the WJ performed the experiment and wrote the manuscript. JL assisted in performing partial experiments. HY, JL, and XC assisted the sample collection. YY assisted the phylogenic reconstruction. GY, YB, and QS conceived the experiments. FW, YB, QS, and GY revised the manuscript. All authors contributed to the article and approved the submitted version.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.


The soil samples were collected eight times. The physicochemical properties were determined for the soil and BioC by analysing the density, pH, surface charge, ash, and organic carbon content. Based on the changes in the structure of the HAs and their quantity in the soils, the chemical properties of the HAs were determined. The maximum BioC dose caused an increase in the content of Corg and HAs. BioC did not influence the humification degree coefficients of the HAs originated from fallow, whereas in the grassland, there were significant changes observed in these coefficient values, indicating that BioC may stimulate and accelerate the humification process of soil HAs. Increasing the BioC doses caused an increase in the soil’s HA content, suggesting an increase in soil sorption capacity. The fluorescence data showed BioC addition to the soil caused an increase in the number of structures characterised by low molecular weight and a low degree of humification.There is limited information regarding the effect of biochar (BioC) on the fertility of fallow and grassland soils, as well as on the properties of their humic acids (HAs). The objective of this study was to evaluate with a 3-year field experiment the influence of BioC on the organic matter (OM) in Haplic Luvisol. BioC (obtained via wood waste pyrolysis at 650 °C) was applied to the soil of subplots under fallow and grassland at doses of 0, 1, 2 and 3 kg m Nevertheless, long-term fallow cannot satisfy requirements at the local level, and moreover, it results in a strong decline in the ecosystem1. Modern agriculture is currently attempting to restore utility value to these soils, and fallow reclamation has become a goal in this context3. Moreover, agriculture is strongly evolving towards sustainable systems and organic farming, thus it is necessary to seek solutions that will not influence soil conditions in a negative manner 4. The answer to this problem could be biochar (BioC)—a solid carbon-rich product generated during waste biomass pyrolysis in a strongly reduced oxygen atmosphere6, which is intended to improve the quality and fertility of soils. This distinguishes it from similar substances like charcoal, which is primarily intended for energy use. Research on the impact of BioC on the physicochemical properties of Haplic Luvisol soil showed that the addition of BioC to fallow and grassland influenced the dynamics of changes in the physiochemical parameters of the examined soils4.Unfavourable changes in the prices of fertilisers and agricultural products in the nineties contributed to the exclusion of some agricultural lands from crop production. In the history of global agriculture, fallows have been known for centuries as an integral aspect of crop rotation. Allowing farmland to fallow can effectively improve the self-rehabilitation of arable soil and solve agroecological problems such as biodiversity loss, soil fertility loss, and over-cultivation10. The literature also includes valuable studies on the effect of BioC on soil organic matter (OM), including the humic and fulvic acids of differently used soils or composting manures12. However, there is no information regarding the potential influence of BioC on improving the fertility of fallow soils, or on the chemical and sorption properties of their humic acids (HAs). According to the literature, BioC may provide a potential soil amendment approach. BioC, due to its high porosity, may improve water absorption, sorption capacity, and nutrient retention, as well as the sorption of organic and inorganic contaminants6. Moreover, BioC contains micro- and macroelements and has high stability in soil13. This component of BioCs plays an important role in determining their potential for environmental use. Clearly, the composition and distribution of soluble OM present in BioC vary significantly according to the type of feedstock, activation, and pyrolysis temperature15 as well as the pH and BioC particle size fraction16. The content of soluble OM in BioC correlates with the BioC volatile matter content and acid functional group density17. In previous study15, the soluble OM obtained from 10 BioC samples produced from different feedstocks was characterized using EEM/PARAFAC  analysis and four fluorescent components: C1, a humic-like component; C2, a protein-/tannin-like component; C3, a fulvic acid-like component; and C4, a terrestrial humic-like component were identified. There are also available, interesting results of research that show that the content of dissolved organic carbon (DOC) extracted from BioC ranges from 435 to 5000 mg kg−1, and that high soluble OM release occurs under high temperature and alkaline environmental conditions18. The authors also identified, using fluorescence analysis, four HA-like substances , one fulvic acid-like substance (C3), and one tryptophan-like substance (C6).There are many scientific reports on the effect of BioC addition to agriculturally cultivated soils on the physicochemical properties of the soils (mainly model studies)20, particularly on HAs. This fraction (HAs) plays a special role among OM compounds by affecting the physical21, chemical22, and biological23 properties of the soil. The acidic character and the nature of negative colloids make HA a valuable buffer compound in soil25 and one of the most highly charged substances among naturally occurring polyelectrolytes26. As a consequence, HAs are responsible for binding cations in the soil, and thus for the circulation and availability of micro- and macroelements. However, HAs are subject to constant structural changes in soil under the influence of various factors. These processes are still poorly understood, although they determine the fate of many other substances in the soil. An analysis of changes in the HA fraction is particularly interesting due to the wide spectrum of HA behaviour in soil ranging from that of fulvic acids to that of humins, i.e. depending on the environmental conditions, HAs are found in highly soluble forms similar to fulvic acids, or as poorly soluble compounds more similar to humins27. Moreover, the HAs in soils vary widely in structural and sorption properties, including their molecular weights , and their number of functional groups, aromatic rings, and aliphatic chains29. Due to such high variability and sensitivity of the HAs to different conditions, it can be assumed that BioC supplementation to soil may also influence the chemical properties of soil HAs. These changes should be monitored, in particular the HA fraction, in relation to the nature of BioC and the method of its production. In the context of pyrolysis and the use of BioC in soil, the higher stability of HAs compared with, for example, fulvic acids, may be crucial. BioC could contain some HAs hidden in the pores such that BioC-derived HAs would increase the HA pool in the soil. This assumption is more likely for HAs than for fulvic acids whose thermal stability is lower (a significant part of the fulvic acids can be destroyed during BioC production). We based these assumptions on our previous studies of the thermal properties of HAs and fulvic acids30 in an N2 atmosphere. Those results showed that fulvic acids were degraded at lower temperatures due to their higher content of O-containing groups and lower content of aromatic structures. This also seems to justify the need to study the changes in HA fraction under the influence of BioC supplementation.The addition of BioC could also have an effect on the pool and chemical properties of the OM in soils32, adding BioC to degraded soils may have significant potential for improving their fertility and productivity by enriching organic carbons, as well as for the protection of plants against diseases. For example, mixing BioCs with Acrisols at a 100 g kg−1 dose increased the pH from 4.9 to 8.7, and this resulted in a 15-fold increase in the DOC concentration (from 4.5 to 69 mg dm−3)31.Further, due to insufficient fertilisation and crop production in monoculture, most agricultural mineral soils are characterised by a low content of OM and significant depletion of HAs. Such environments require additional supplementation with organic carbon compounds. According to different sourcesSoil degradation due to land mismanagement is a major global concern that threatens economic and rural development. Therefore, the aim of this work was to investigate the effect of BioC addition to degraded soils . This goal was realised by examining (1) the effect of BioC on the OM sorption properties of soils from fallow and grassland, and (2) BioC’s influence on the HAs of these soils as a function of BioC dose and the 3-year duration of the experiment. By examining the dynamics of changes in the content and properties of OM, and its most important fraction, HAs, we would like to examine the suitability of BioC to improve the quality of degraded soils.The physicochemical and chemical characteristics of the soils and BioC, as well as selected chemical properties of the HAs isolated from the soil and BioC are shown in Table org, A, pH, and Q, were presented in detail previously4. Briefly, soils were characterised by a typical d value for mineral soils ≈ 2.60 g cm−3, and by a relatively low content of Corg and a high content of A. The pH of the soils was weakly acidic. The examined soils were characterised by low Q values, indicating a low content of organic structures dissociating to the negative surface charge (mainly carboxylic and phenolic groups). The HAs obtained from fallow and grassland were characterised by high QHA values . The d value of BioC was typical for organic materials (1.46 g cm−3), moreover, the BioC contained a high content of OM, which was expressed as Corg. The pH of BioC was alkaline. This material was also characterised by a high Q value, which indicated its favourable sorption properties.The properties of soils and BioC, such as the d, C2/6 values were similar for the HAs originated from the two studied soils, suggesting a similar ratio of lignin-type compounds resistant to humification to the structures with a high humification degree. The ΔlgK reached values of 0.83 and 0.86 for HAs isolated from grassland and fallow, respectively, indicating a low degree of HA humification (Kumada’s classification for low humification degree of HAs: ΔlgK = 0.8–1.1)33. Slightly higher ΔlgK values obtained for the grassland HAs compared with the fallow suggested a higher content of less humified compounds, such as cellulose, hemicellulose, and lignin34.The results of our studies showed that the E2/6 parameter of the HAs fraction isolated from BioC indicated a low content of lignin-type compounds31.The ΔlgK of HAs isolated from BioC reached a value of 0.54, suggesting the presence of highly humified compounds, in comparison with soil HAs (Kumada’s classification for high humification degree of HAs: ΔlgK < 0.6). A particularly low value for the E35. High temperatures (600 °C and higher) can result in a decrease in CEC  and process gases (15–35% of the input energy), and the process was based on autothermal roasting without the use of additional energy, catalysts, or chemical additives. BioC produced from wood waste (FLUID technology) showed a qualitative increase in physical and chemical properties in comparison with untreated biomass in relation to the moisture, quantity of carbon element, volatile matter, and calorific value.The BioC properties presented above largely depended on the conditions of the pyrolysis process, as well as on the feedstock. The BioC used in our experiment was obtained via wood waste (coniferous and deciduous) pyrolysis at a relatively high temperature of 650 °C. The first rapid stage of this process (to 200 °C) could be attributed to the evaporation of moisture and light volatiles, the second one (200–500 °C) to devolatilisation and decomposition of hemicelluloses and cellulose, and the last one to the degradation of lignin and other OM with stronger chemical bondsorg content of the soils  forming insoluble salts with organic ligands of the BioC43.After increasing the C−1. This is a relatively low value, which resulted from the conditions of the pyrolysis process. High temperature of the process (650 °C) caused the breakdown of cellulose, hemicellulose, and lignin. As a consequence, the content of volatile matter markedly decreased, but the ash content increased. This was observed due to separation of volatile fractions into low-molecular-weight liquids and gases, which evaporated from the BioC40. Studies on the effects of pyrolysis temperature on the physicochemical properties of different BioCs44, showed that volatile matter ranged from 340 to 540 g kg−1 in the BioCs obtained at 300 °C as well as from 60 to 190 g kg−1 in BioCs produced at 600 °C. Because BioC lability was found to be strongly controlled by the relative amount of a more aliphatic and volatile component, the measurement of volatile compound content may be a convenient predictor of BioC carbon (C) longevity. The lower the level of volatile components, the more stable the BioC in the soil, and consequently, the longer the impact on soil properties45. This fact was confirmed in our research: the most significant changes were observed in the third year of the experiment. From an environmental point of view, the volatile matter content of the BioC is an important factor affecting its agronomic value as a soil amendment. Chars that are rich in volatile matter content  would not be good soil amendments because they overstimulate microbial activity or immobilise the nitrogen (N) available to plants in the short term. What is more, soil amended with BioC having high volatile matter content showed a dramatic decline in soil respiration45. The relatively low content of volatile compounds in the BioC used in our research seemed to be safer for the environment. The literature shows that the release of volatile compounds into water and soil can have adverse effects on plants and microorganisms47.The stability and chemical quality of the BioC used in our experiments also strongly depended on its content of volatile organic compounds. Their amount in the analysed BioC was approximately 150 g kg49. The presence of the above structures determines both the sorption and buffer capacity of soil systems48. Surface functional groups dissociating at pKs 7.0–9.0 were dominant, revealing the higher content of phenolic and hydroxylic groups compared with carboxylic structures.The data obtained from potentiometric titration allowed for an estimation of the distribution functions of apparent surface dissociation constants in the fallow and grassland amended with the BioC. These characteristics illustrated quantitative and qualitative changes in surface functional groups. The exemplary distributions hare shown in Fig. 50, which demonstrated that BioC has a long-term effect, noticeable only a few years after its application to the soil. The effect of carboxylic groups of BioC is interesting because BioC production by biomass pyrolysis results in the partial degradation of oxygen-containing functional groups52. No similar changes were observed in the distribution of dissociation constants of grassland amended with BioC  did not cause significant changes in the apparent surface dissociation constants for either the fallow or the grassland Fig. A,C. In torgHA) demonstrated that BioC amendment also influenced the total content of the HAs both in fallow and grassland. The influence of BioC doses on the content of HAs in the soils expressed as a concentration of organic carbon in the HAs extracts is shown in Fig. orgHA in Fig. −2 BioC) by approximately 15%—both in fallow and in grassland. The observed decrease in the HA content in some terms most likely resulted from the intensified mineralisation process of native organic compounds54.The analyses of organic carbon concentration in the extracts of HAs 58. According to literature57, the positive priming from BioC may also be related to relatively greater lability of native organic C and its lower protection in soil with clay content < 200 g kg−1 (the clay content in Haplic Luvisol used in our experiment was approximately 70 g kg−1). The stabilisation of SOC and BioC C through organo-mineral associations is expected to be related to the soil clay content. In soils containing > 200 g kg−1 clay, stabilisation or decrease in native SOC mineralisation in the presence of BioC may occur through BioC induced organo-mineral interactions.Another aspect of principal importance is the priming effect that BioC may exert on native soil organic carbon (SOC) and the converse, i.e., the priming effect of SOC on BioC C. This is important, especially if BioC is used for C storage capacityHA) obtained from fallow, grassland, and BioC in the pH range of 3.0–9.0  as compared with that in the grassland. On the other hand, the continuous increase in E2/6 values of the grassland HAs as a function of the 3-year duration of the experiment may be related to the addition of further portions of lignin-type compounds, originated from fresh biomass, which were present on the grassland surface.It should be noted that the value of Els Table . Hence, th month of the experiment), while the BioC addition reduced the is decrease in degree of humification as a function of time, which was expressed in lower and more consistent ΔlgK values in months, as compared to the control soil.Data obtained from spectrophotometric analyses allowed calculation of the ΔlgK parameter describing the humification degree of the HAs. Changes in the above parameter for HAs isolated from fallow and grassland amended with BioC are provided in Fig. 2/6 . Moreover, literature indicates that, the humification process of the OM is stimulated mainly by the presence of microorganisms and plant residues59. Therefore, the lack of plant cover suggests lower biological activity of the soil and, consequently, a lower intensity of humification processes. The reduction in ΔlgK (i.e. the increase in the humification processes) in relation to the control sample, as well as the stabilisation of ΔlgK values as a function of time after BioC supplementation, can be explained for grassland in two ways. Firstly, there is possibility that BioC may stimulate and accelerate the humification process of soil OM41. Moreover, BioC supplementation resulted in the direct introduction of strongly humified HAs , as well as an increase in the ratio of HAs to fulvic acids.Indeed, humification processes are chemical and biochemical transformations, where microorganisms play a key role in the decomposition of OM into simpler organic structures. In these processes, the organic substrate is recolonised by microorganisms that degrade the residual sugars, cellulose, and hemicellulose, and increase the rate of polymerisation of the organic compounds to the humic substancesThe results of the fluorescence analysis supplemented the influence of BioC on the HA structure in terms of changes in fluorescent groups. The obtained EEM data showed the presence of one local maximum for fluorescence (marked as α)—the results are shown in Fig. 66. The activity of the α area was visible in all studied HA samples at excitation/emission wavelengths of 305–330 nm and 420–430 nm. According to the literature69, the α region was related to terrestrial humic-like substances derived from lignin and may reflect simple structural components of low molecular weight, low content of conjugated and aromatic groups, and low degree of humification. The highest fluorescence intensities (FIs) of this region were observed for HAs of grassland amended with BioC (3 kg m−2) during the first month of the field experiment . At the same time, it should be noted that together with such HAs, fulvic acids can be formed. This fraction is also characterised by a low molecular weight and a low degree of humification70. This could be important information for future research that includes the fulvic acid fraction. We suppose that this fraction may be important regarding humification products formed in a relatively short time. The β signals were placed at ex/em: 370–380 nm/480–490 nm, with the strongest signal for HAs obtained from the grassland during the first month of the experiment with and without the addition of BioC . The BioC addition caused an increase in the number of highly humified structures in the HAs obtained from grassland during the first year of the experiment, and from fallow in the last year of the experiment.The γ area was observed for HAs obtained from grassland with BioC addition during the first year of experiment Fig. D, as wel74  and included 4 fallow and 4 grassland subplots, each of 20 m2, with a separation of 1 m between the plots. Figure The studies were conducted on a Haplic Luvisol (IUSS Working Group WRB)Festuca pratensis Huds., Dactylis glomerata L., Poa pratensis L., and Lolium perenne L. The fallow surface had been left unseeded after being tilled to a depth of 15 cm and harrowed for 10 years prior to the experiment. During the research, the fallow plots were maintained without plants. All plots, before and during the experiment, were fenced to ensure that animals were not able to enter.The grassland was established at least 35 years ago and was managed by cutting the surface covered with various grass species, such as: −1. The dry product was uniformly applied to the surface of the subplots at densities of 0 (control), 1, 2, and 3 kg m−2 in June of 2013. The BioC doses were selected based on our previous studies on the impact of BioC on the physicochemical properties of Haplic Luvisol soil under different land use4. In the case of fallow, BioC was mixed to a depth of 0–15 cm (surface layer) using a rototiller, and it was left on the surface in the case of grassland76. To avoid BioC loss, the plots were initially wetted. Soil samples were collected (in triplicate) from the surface layer (0–15 cm) from each fallow and grassland plot, during the summer, autumn, and spring periods  during the 3-year experiment: two times in the first year (starting from July—the first month of the experiment), three times in the second year, and three times in the third year (the last one in November which was the 28th month of the experiment). The collected material was dried at 25 °C, sieved through a 1 mm sieve, and then grounded.The BioC used in the experiment was obtained via wood waste (coniferous and deciduous) pyrolysis at 650 °C (Fluid S.A.); the pyrolysis time was 15 min, and the heating rate was approximately 3 K s2O and 0.01 mol·dm−3 CaCl2 solution . The ash content (A) was determined as the loss of BioC calcination in a muffle furnace  at 550° C for 5.5 h. The organic carbon content (Corg) was analysed using a TOC analyser . The negative surface charge (Q) was calculated at pH 9.0 from potentiometric titration curves recorded for aqueous suspensions of soil or BioC (5 g kg−1) titrated with 0.1 mol·dm−3 NaOH (based on 1 mol dm−3 NaCl) in a pH range of 3.0 to 10.0 using the automatic titrator TitroLine 7000 26.Selected physicochemical and chemical properties were determined for soil and BioC characterisation. The density (d) was determined using the pycnometric method ; the pH was measured in Horg content in the soils using the same method as for the control soil characterisation. The distribution functions of the apparent dissociation constants of the functional groups were determined for the OM in soils amended with BioC based on the potentiometric titration curves obtained for the mixture of soil with BioC in the pH range of 3.0–10.0 using the same conditions and calculations as for the characterisation of the initial materials.The effect of BioC addition on the OM of the soils was investigated with an analysis of the changes in C77. The changes in the HA organic carbon concentration (CorgHA) were analysed in the HA alkaline extracts using a TOC analyser . The UV–Vis spectra were recorded for HA solutions (concentration = 40 mg dm−3) in the wavelength range of 200–800 nm . From the obtained spectral data, the ΔlgK parameter78 (describing the HA humification degree) and E2/6 coefficient (describing the ratio of lignin-type compounds to the content of substances with a high degree of humification) were calculated as follows:x is the absorbance measured at a specific wavelength.HAs were isolated from the BioC, control soil, and soil amended with BioC using an alkaline extraction method proposed by the International Humic Substances Society−3 NaOH, and the absorption curves were promptly drawn in the region of 220–700 nm78. These curves were expressed as lg K-λ curves of 10 g dm−3 HA solution, where K and λ were the optical density and wavelength, respectively.The absorption spectra of HAs were determined in dissolved 0.025 mol dmHAs were determined for HAs of soils amended with BioC based on the potentiometric curves in the pH range of 3.0–9.0 using the same titration conditions as in the case of the characterisation of the initial materials’. The values of ΔlgK, E2/6 as well as the QHAs for the HAs of the control soils and for the BioC were attached to the data describing the research materials.The changes in Q3 of deionised water. The pH of the solutions were adjusted to 7.5 ± 0.1 using 0.1 mol dm−3 NaOH. The emission-excitation fluorescence 3-D matrices (EEM) were recorded with a scan speed of 30,000 nm min−1 using the Hitachi F-7000 FL luminescence spectrometer . The emission wavelength was scanned from 300 to 600 nm, the excitation wavelength was raised sequentially in 5 nm steps in the range of 250–500 nm. The analyses were preceded by fluorescence calibration using quinine sulphate at λex = 350 nm and λem = 450 nm. Spectral correction was performed using rhodamine B. The EEM data were processed into contour maps using Surfer software 70.The HA solutions for the fluorescence measurements were prepared by dissolving 40 mg of each lyophilised sample in 1 dmStatistical analysis was performed using Statistica 12.0 : a one-way analysis of variance (ANOVA) and a post hoc analysis .The results obtained suggested that the BioC application seemed to have a subtle influence on soil characteristics, leading to changes in the OM, including HAs. The addition of BioC had various influences on differently cultivated soils—fallow and grassland.org content which ranged for different months from 3 to 20% and from 15 to 25% as compared to control values in the fallow and grassland, respectively. The Corg content in both soils after the addition of BioC remained constant throughout entire 3-year experiment, indicating the high stability of organic compounds introduced with BioC.The addition of increasing BioC doses caused an increase in C2/6) for HAs originated from this soil. Similarly, the addition of BioC to the fallow did not change the humification degree (ΔlgK) of this soil’s HAs. In the case of grassland, a gradual increase in the E2/6 values of the HAs was observed during the 3-year experiment, indicating an increase of lignin-type compounds in the structure of the soil’s HAs. The application of BioC to the grassland stimulated and accelerated the humification process of this soil’s HAs. Moreover, the content of HAs in both soils increased significantly with an increase in the BioC dose at each term of the experiment, suggesting an increase in soil sorption capacity.BioC addition to the fallow did not significantly influence the parameter for the content of lignin-type compounds (EBased on the EEM data analysis, we can conclude that BioC addition to the soil caused an increase in the number of structures characterised by low molecular weight and a low degree of humification. Furthermore, its addition caused an increase in the structures characterised by a high degree of humification in the HA structures obtained from grassland in the first year of the experiment and from fallow in the last year of the experiment.The results of this study support the thesis that the addition of BioC to degraded soils may improve their fertility.