Temperate phages are viruses of bacteria that can establish two types of infection: a lysogenic infection in which the virus replicates with the host cell without producing virions, and a lytic infection where the host cell is eventually destroyed, and new virions are released. While both lytic and lysogenic infections are routinely observed in the environment, the ecological and evolutionary processes regulating these viral dynamics are still not well understood, especially for uncultivated virus-host pairs. Here, we characterized the long-term dynamics of uncultivated viruses infecting green sulfur bacteria (GSB) in a model freshwater lake . As no GSB virus has been formally described yet, we first used two complementary approaches to identify new GSB viruses from TBL; one in vitro based on flow cytometry cell sorting, the other in silico based on CRISPR spacer sequences. We then took advantage of existing TBL metagenomes covering the 2005–2018 period to examine the interactions between GSB and their viruses across years and seasons. From our data, GSB populations in TBL were constantly associated with at least 2-8 viruses each, including both lytic and temperate phages. The dominant GSB population in particular was consistently associated with two prophages with a nearly 100% infection rate for >10 years. We illustrate with a theoretical model that such an interaction can be stable given a low, but persistent, level of prophage induction in low-diversity host populations. Overall, our data suggest that lytic and lysogenic viruses can readily co-infect the same host population, and that host strain-level diversity might be an important factor controlling virus-host dynamics including lytic/lysogeny switch. Recent advances in metagenome sequencing have enabled high-throughput exploration of the virosphere, leading to a >200-fold increase in viral genomes available in databases, and uncovering >1,000 new genus-level viral groups . While tFor viruses infecting bacteria and archaea, viral infection dynamics exist along a spectrum from highly lytic to lysogenic and/or chronic ; highly The existence of such diverging hypotheses, both with supporting data, reflects the complexity of virus-host interactions and the limits of our current understanding. In particular, the majority of the hypotheses put forward so far have been based on community-wide measurements, which average out differences between individual viruses and host populations , 9. In aIn this study, we focus on uncultivated viruses infecting green sulfur bacteria (GSB) in a model freshwater lake  sampled from 2005 to 2018 by the North Temperate Lakes Microbial Observatory. GSB are anoxygenic photoautotrophic bacteria that can form massive blooms in illuminated, low-oxygen, sulfidic waters, and play a central role in global carbon and sulfur cycling –25. BecaTo investigate viral infection dynamics of GSB in TBL, we first used two complementary approaches to identify viruses infecting GSB. We next followed newly-established virus-host pairs across multiple years and seasons using bulk metagenomes covering the 2005–2018 period to evaluate patterns of infection rate and virus-host interactions at the population level. Finally, we highlight through direct observation and theoretical modeling that host strain-level diversity may be a critical parameter driving these virus-host dynamics.Chlorobaculum tepidum, see Methods). Then, the relative abundance of GSB in TBL was quantified using FACS at individual depths (depth-discrete samples) across all sampling dates in 2018  followed by Fluorescence Activated Cell Sorting (FACS), and the other aimed to collect the entire microbial community by sampling across the hypolimnion, i.e., the bottom layer of the lake . The overwhelming majority of GSB cells were found right below the oxygen barrier/chemocline . Each sampling date included 1 or 2 nearly complete GSB genome bins , targeted metagenomes were sequenced from different depths across 2018, highlighting differential distribution patterns for GSB-A and GSB-B see Fig. . Specifiave bciD  nor cruBuB genes . It is uA third bin was routinely found in the 2018 targeted metagenomes, however it was estimated to be only 20% complete, i.e., it included very few to none of the expected core bacterial genes. This third bin was clustered next to the other two GSB bins when included into an rpoB phylogeny, suggesting it also represented a GSB population Fig. . A similChlorobium strains, or technical biases associated with isolation or prophage identification.To identify viruses infecting GSB, we first used VirSorter to detect viral contigs from the 2017 and 2018 GSB-targeted metagenomes, and excluded putative contaminants based on coverage patterns across replicates (see Methods). Across all 14 sampling dates in 2018, we recovered 43 nonredundant predicted GSB viral contigs  Table . While mPutative GSB viruses were also recruited from all available TBL metagenomes from 2005 to 2018 by matching spacers from the CRISPR arrays associated with GSB-A and GSB-B to predicted viral contigs (see Methods). This yielded a total of 534 contigs, which were subsequently clustered into viral OTUs (95% ANI and 85% AF) to produce 45 nonredundant viral contigs (34 of the contigs were >10 kb). Out of the 45 contigs, 6 matched viruses recovered through GSB-targeted metagenomes (see above). Overall, combining results from the flow sorting method (targeted metagenomes) and the CRISPR spacer-matching method resulted in 50 nonredundant GSB viruses. Only one putative GSB viral sequence was described in the past, identified as a virus infecting GSB based on metagenomic coverage, however it bears no similarity to the GSB viruses identified here, either in flow sorting of from CRISPR .Caudovirales order  were connected to the main component of the network, confirming that these are likely members of the er Figs. . HoweverThese two clusters of GSB viruses showed different patterns based on shared gene content and host. In VC-2, three genomes assembled from the 2018 targeted metagenomes were clustered with a 34–59% shared gene content; of the three, one was associated with GSB-A, while the two others were associated with GSB-B, based on genome binning and co-detection in read mapping analyses. Based on their shared predicted gene content, the GSB viruses in this cluster appear to be recently diverged and co-diversifying each with a specific host population Fig. .Overall, our identified GSB viruses are distinct from all known viruses including one putative GSB virus from ), may reMost (26) of the 45 viral OTUs detected in 2005–2013 based on CRISPR matches were not detected in 2018, either in targeted or bulk metagenomes Figs. , S6. A tThe fact that different GSB viruses are identified through targeted metagenomes and CRISPR spacer matching highlights how these two approaches are complementary, as they reflect different infection status Fig. . While Csox cluster for thiosulfate utilization as a well-known example [Auxiliary metabolic genes (AMGs) are host genes found in bacterial viruses that can modulate host cell metabolism during infection, usually to increase the efficiency of viral replication. This phenomenon has been observed in many bacteria, including e.g., cyanobacteria, where some viral-encoded proteins are used during infection to maintain host cell photosynthesis capacity . In GSB, example . It is tOverall, we found no clear evidence for AMGs in any of the TBL GSB viral genomes. Specifically, none of the sequences identified as a putative GSB virus through targeted metagenome or CRISPR match included a predicted gene that could confidently be linked to a cellular metabolism. Since most (>60%) of putative GSB virus genes were not or poorly annotated however, it is possible that some GSB viruses do encode genuine AMGs, but further experimental characterization of these unknown genes will be required to identify these.Mapping reads from individual bulk metagenomes to the new GSB genomes and GSB viruses assembled from targeted metagenomes enabled a more detailed investigation of the temporal dynamics of GSB and their viruses in TBL. These data provide a unique look at virus-host dynamics, as these data cover more than 10 years, and represent one of the longest running datasets currently available to investigate viral–host dynamics in natural systems. Based on read mapping, GSB-A was present in all years dating back to 2005, and was, until 2018, the dominant host population Figs. , S7. ItsIn contrast, GSB-B was overall less abundant, with reliable coverage (>10×) only achieved in 2005, 2018, and some samples in 2007. While it was not the dominant GSB population for most of the years covered by our data, it was clearly more abundant than GSB-A in 2018 Figs. , S5. SimAt the community level, a mix of persistent viruses (repeatedly detected across seasons and years) and transient viruses (detected in only one or a few time points) have been reported in marine systems –48 and iAs previously reported in ref. , GSB-A wIn addition to estimating microdiversity within GSB hosts and their associated viral populations each year, we also looked at the turnover between populations each year by calculating the proportion of SNPs for which the dominant allele changed between years. This was calculated similar to our standard SNP calculations, but instead of looking at overall SNPs density across all samples, pairwise comparison of years was computed with samples per year pooled. By looking at turnover in the GSB populations, we can estimate how similar (or dissimilar) a given population is compared to the population in the year before or after. In GSB-A, we see a similar pattern as the within-sample nucleotide diversity, with higher SNP turnover between 2005 and 2007, and between 2013 and 2018 Fig. , suggestAlthough GSB-A and GSB-B grow under similar ecological conditions and harbor similar genomes Table , these tIn contrast, for populations with higher levels of strain diversity such as GSB-B, there is likely an existing pool of strains with potential for resistance. For this population, instead of stable prophages, we observe either a “rare” prophage, or a rapidly changing lytic virus that would be consistent with ongoing kill-the-winner/Red Queen type dynamics. Hence, in higher-diversity host populations, the most successful strategy for viruses could be lytic or short lysogenic infection cycles, leading to more replication events and a more diverse viral population. Temperate viruses infecting these higher-diversity host populations, while less successful (in terms of relative abundance and infection rate), could still be stably associated with a subset of the host population as observed here in the GSB-B virus, CV-1-4.2.To examine the potential for these scenarios to occur under different conditions, we adapted an existing theoretical model  to consiy-axis). However, this final lysogen rate was also dependent on induction rate  and ~50% infection rate Fig. . Here weIn this study, we characterize viruses infecting GSB in a model freshwater lake. By leveraging the unique autofluorescence of GSB cells, we sorted GSB cells using FACS, and sequenced these “targeted metagenomes” to identify GSB-specific viruses. These targeted metagenomes allowed us to observe cell-associated viruses, and ecologically relevant ongoing infections. Overall, this newly developed method recovered novel viruses with a host context at high (population-level) resolution. These new host-contextualized viruses in turn enabled a re-analysis of time series bulk metagenomes to investigate virus-host interactions across seasons and years in a model freshwater lake. For one virus persistent through multiple years (CV-1-33 infecting GSB-B), we observed temporal dynamics and microdiversity patterns consistent with a “kill-the-winner” dynamic. However, for a closely related host population, viruses persisting throughout multiple years seem to be instead temperate and consistent with a “piggyback-the-winner” dynamics (CV-1-51 infecting GSB-A). A theoretical model of these infections suggest that host population diversity may be a critical factor driving these virus-host interactions by influencing the potential for resistance in each host population. “Piggyback-the-winner” strategies would be more successful when prophages with low induction rates are able to spread through low-diversity host populations and infect all members of the dominant strain, while “kill-the-winner” dynamics would more frequently occur in high-diversity host populations which include a subset of resistant cells. Consequently, the optimal latency time decision of viruses would in turn depend partly on the host population diversity, with long-latency prophages favored in low-diversity host populations. This would be consistent with the link between lysogeny and host life-history traits, as lysogeny-associated traits such as “pathogenicity” or “boom-and-bust” life cycles are also associated with population bottlenecks, and could also explain the imperfect linkage between lysis-lysogeny rate and broader ecological parameters such as host abundance, nutrient availability, and growth rate. While this study focuses on one environment, two specific host populations, and their “in-cell” viruses, it would be of interest to verify if the general patterns observed also hold true for other GSB populations in other lakes, and for other bacterial populations with varying growth cycles and dynamics.Sphagnum mat that supplies large amounts of terrestrially derived organic matter to the lake, leading to darkly stained water and greatly attenuated light penetration. The lake mixes twice per year  but thermally stratifies strongly in the summer. It has a maximum depth of 7 m, surface area of ~11,000m2, and a mean pH of 5.1 [https://lter.limnology.wisc.edu/researchsite/trout-bog); these yearly temperature changes along with other major biogeochemical parameters can be seen in Fig. TBL is located in Vilas County, Wisconsin, USA, and is surrounded by a H of 5.1 . DissolvH of 5.1 , https:/Two different types of samples were collected during June-October 2018: (1) depth-integrated water samples were collected onto filters (see below) from the hypolimnion layer (as defined by the temperature gradient) at 14 different time points, and DNA was purified from these filters using the FastDNA Spin kit for Soil , with minor modifications to the kit protocol, as described previously ; (2) depFor the depth-integrated samples , one library from each sample was prepared following standard KAPA kit protocol. 10 ng of Genomic DNA was sheared to 300 bp using the Covaris LE220 and size selected with SPRI using TotalPure NGS beads . The fragments were treated with end-repair, A-tailing, and ligation of Illumina compatible adapters  using the KAPA Standard Library Creation kit (KAPA Biosystems) and five cycles of PCR was used to enrich for the final library.g for 1 h at 10 °C, inverted and centrifuged at 7 × g for 5 s to remove supernatant, and amplified by MDA at 45 °C for 16 h using EquiPhi29 DNA polymerase (ThermoFisher Scientific) following the protocol described in ref. [For the depth-discrete samples , Nextera XT v2 (Illumina) sequencing libraries were generated from 5,000 sorted cells following multiple displacement amplification (MDA) . Five li in ref. . Paired-C. tepidum. Briefly, the influx cell sorter (BD Biosciences) was prepared following the protocol for single-cell genomics outlined in [Benchmarking for this protocol was performed on cultures of lined in . Howeverlined in , 57, wheFor depth-discrete water samples (hereafter “targeted metagenome”), each dataset was subsampled to 60 million total reads per sample using BBMap reformat function . For eacTBL GSB genome bins were annotated using Prokka . Genes To identify potential viral contigs, contigs from all 14 2018 co-assemblies of sorted cells and all bulk metagenome assemblies were analyzed by VirSorter 1.0.5 . BrieflyTo identify contigs from putative GSB viruses using matched CRISPR spacers, all metagenomes were first processed with the Crass assembler (v0.3.12) , to broaFor taxonomic affiliation, all viral contigs identified (including the ones not associated with GSB) with a length ≥10 kb were pooled with reference genomes from NCBI RefSeq , along with viral sequences from the IMG/VR database with a predicted GSB host , prophagDepth-integrated samples (referred to as “bulk metagenomes” here) were previously taken from TBL in 2005, 2007–2009, and 2012–2013 . In ordeSNPs were identified using Anvi’o v5.5 . Only SNp  and cost (growth cost) as additional parameters, and includes the following nonlinear ordinary differential equations:The virus-host model used here was based on , with p b , K , φ , d , β , m , and n . Additional parameters are μR , μS , p , r (effect of resistance on cell growth), and cost (effect of lysogeny on cell growth). Cell death (unrelated to phage infection) was kept identical across all cell types. The adsorption of virus particles to susceptible and lysogenic cells was also kept identical, with the latter being an evolutionary dead-end , and the adsorption to resistant cell was considered as null .S, R, L, and V denote the densities of susceptible cells, resistant cells, lysogenic cells, and virus particles per ml, respectively. N denotes the total density of cells, i.e., S + R + L. The parameters similar to  include −6 to 1 and the initial proportion of lysogenic cell from 0 to 100%. The initial number of cells was set at 1 × 104 ml−1 based on the flow cytometry counts of GSB for the first samples in our 2018 time series, while the maximum carrying capacity K was set at 3 × 106 based on the maximum count of GSB throughout the 2018 time series (see above). The initial number of viral particles was set at 1 × 104 ml−1 . Additional simulations were run with an initial number of viral particles of 1 × 105 ml−1 and 1 × 106 ml−1, and the same dynamics and patterns were observed as the ones presented in Fig. The two parameters varying between computations were n (induction rate) from 1 × 10β = 50, φ = 6.7 × 10 − 10 ml h−1, and m = 1/48 h−1, which correspond to standard phage infection dynamics. This virion decay rate of ~0.015 is within the range of values previously observed in freshwater systems, although decay rate has not been measured for GSB phages specifically [p was set at 1 × 10−5 h −1 corresponding to ~ 0.05% of prophage loss per generation at the maximum growth rate of 0.02 h−1. Mutation rate from susceptible to resistant (μR) and from resistant to susceptible (μS) was arbitrarily set at 1 × 10−6 to ensure a constant pool of both susceptible and resistant host cells in the system. Finally, the adjusting parameter for lysogenic growth rate was set at 0.99 to reflect the added energetic cost of the integrated prophage, and the adjusting parameter for resistant cells growth rate was set at 0.90 to allow for the establishment of typical prey-predator dynamics for high induction rate .No measurement could be made or obtained from the literature for the other parameters, which were thus set to standard values as follows: Burst size, adsorption rate, and viral decay were set based on , i.e., βifically , 81. Pro−6 to 1 evenly distributed on a base-10 log-scale) and initial proportion of lysogenic cells (0–100% by increment of 5%) for 6570 h, i.e., ~9 months, corresponding to a full seasonal bloom of GSB as observed in our time-series. Since we do not have any data about host cell and virus:host dynamics during the time between blooms, i.e., late fall, winter, and early spring, for which we do not have samples, we opted to not run the model across multiple years. Infection rate and resistant host density data were compiled from the second half of each simulation , i.e., after the initial growth of the GSB population and once the bloom was established. For Fig. Simulations were run separately for a combination of lysis rate (30 values from 1 × 10Supplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6


This study aimed to compare drug costs and healthcare costs of a 1 year adjuvant course with intravenous biosimilar trastuzumab vs. subcutaneous reference trastuzumab in HER2-positive breast cancer from the Belgian hospital perspective. Our simulation is based on the methodology used by Tjalma and colleagues, and considered costs of drugs, healthcare professional time and consumables. We calculated intravenous drug costs for different body weights, and computed drug costs and healthcare costs to treat 100 patients with either trastuzumab formulation, assuming a binomial body weight distribution in this sample. Scenarios were run to account for drug discounts and intravenous vial sharing. Drug costs amounted to €1,431,282 with intravenous biosimilar trastuzumab and €1,522,809 with subcutaneous reference trastuzumab for a sample of 100 patients in the base case analysis. When healthcare professional time and consumables were also considered, healthcare costs with intravenous biosimilar trastuzumab were similar to those with subcutaneous reference trastuzumab. Differences in healthcare costs between intravenous biosimilar trastuzumab and subcutaneous reference trastuzumab depended on the level of discounts on these formulations and on intravenous vial sharing. Our case study demonstrates that comparing costs of intravenous vs. subcutaneous formulations is complex and multifactorial, and entails more than a simple cost comparison of products. Trastuzumab has played, and continues to play, a pivotal role in the standard first-line treatment of HER2-positive breast cancer for approximately two decades. Initial approval was based on the significant overall survival advantage demonstrated in key clinical trials in both the metastatic ,2,3 and A drug cost comparison at 2017 ex-factory prices in Belgium has been performed for the IV and SC formulations of reference trastuzumab for patients in different weight categories . The calA biosimilar is a biological medicine that is highly similar to another already approved biological medicine (the “reference medicine”) and does not show clinically meaningful differences from the reference medicine with respect to pharmaceutical quality, efficacy, and safety . Several®, Roche) as adjuvant treatment for one year in women with HER2-positive breast cancer from the hospital perspective in Belgium as an example to show the multifactorial character of an at-first-sight simple comparison. Our study is based on the methodology used by Tjalma and colleagues [The aim of this case study was to compare drug costs and healthcare costs of IV biosimilar trastuzumab vs. SC reference trastuzumab  ranged from €17,858 for a patient weighing 87.5 kg to €10,244 for a patient weighing 50 kg see . In the We next determined the difference in healthcare costs  between the IV and SC formulations. This calculation took into account that the IV trastuzumab administration was previously estimated to cost €907.20 per course more than SC administration in terms of healthcare professional time costs and consumables costs . Figure When calculated for a sample of 100 patients, the difference in drug costs between the IV and SC formulations amounted to €91,527 see . When alThis study has simulated drug costs and healthcare costs for a 1 year course of adjuvant treatment with either IV biosimilar trastuzumab or SC reference trastuzumab in HER2-positive breast cancer patients in Belgium. Our results indicated that the cost difference between IV and SC formulations depends on patient body weight, drug discounts and IV vial sharing.In our base case analysis, drug costs were less for IV biosimilar trastuzumab for a patients weighing less than 75 kg. The median weight of women with breast cancer is invariably <75 kg and has ranged from 64 to 72 kg in European studies comparing IV and SC reference trastuzumab administration ,18,19,20When considering healthcare costs, our base case analysis took into account that IV administration is associated with more costs related to healthcare professional time and consumables than SC administration, in addition to differences in drug costs. However, savings in healthcare professional time and consumables with SC administration might not be as high when trastuzumab is given in combination with chemotherapy. When trastuzumab is administered in combination with chemotherapy, this is usually for the first 6–8 cycles of 18 cycles during adjuvant therapy. During these 6–8 cycles, there are potential cost savings with respect to healthcare professional time and consumables with IV trastuzumab administration by piggy backing on the costs that must be applied for IV chemotherapy administration during concurrent or sequential administration. The combination of trastuzumab with chemotherapy is usual practice (94%) during adjuvant therapy across German hospitals , whereasMultiple studies have reported that SC reference trastuzumab administration is associated with less indirect costs related to productivity loss than IV administration ,19,20. OWhen we applied healthcare cost estimates to a sample of 100 patients, lower drug costs with IV biosimilar trastuzumab as compared to SC reference trastuzumab offset higher costs of healthcare professional time and consumables in our base case analysis. Also, we ran scenario analyses accounting for drug discounts and for the re-use of IV vial leftovers. We believe that these scenarios more accurately reflect market and clinical practices in Belgium, even though the related input parameters are associated with more uncertainty and resulting cost difference estimates are illustrative rather than exact. In terms of generalizability to other healthcare systems, healthcare cost differences between these trastuzumab formulations of course depend on the difference between the drug procurement cost and reimbursement rate, on local healthcare professional and consumable costs, and on the hospital or retail setting in which IV and SC formulations are typically provided.Our results are in line with those of an Italian study , which fWe hope that our case study contributes to a more differentiated view on the difference between IV and SC formulations beyond the bare price of the products alone. Indeed, we acknowledge that other factors may also play important roles like the business models of hospitals and the earning system of physicians. A hospital that is short in IV administration capacity, and gains limited earnings from IV administrations, may like to avoid investments to expand such (expensive) capacity. On the other hand, if physician reimbursement for IV administration is higher than for SC administration, then it will be attractive for physicians to favor the former. In a number of countries, parenteral drugs are increasingly being administered outside the hospital, closer to where patients are living. Such initiatives are more dependent on the availability of SC formulations.There are a number of limitations in our study. The estimate of cost savings related to healthcare professional time and consumables with SC trastuzumab administration related to 2017 , while dFew studies have explored cost differences between IV biosimilar trastuzumab and SC reference trastuzumab . More re®) was €276.87 per 150 mg vial and that for SC reference trastuzumab (Herceptin®) was €846.01 per 600 mg vial [Calculations of drug costs for IV biosimilar trastuzumab vs. SC reference trastuzumab were conducted in the same manner and following the same methods as reported for the comparison of IV vs. SC reference trastuzumab in the study by Tjalma and colleagues . Drug co mg vial .Next, we compared healthcare costs for IV biosimilar trastuzumab vs. SC reference trastuzumab at the previously defined different patient body weights (see above) by taking into consideration potential savings through SC use that have been previously estimated by Tjalma and colleagues . They esn = 7); 84.0 kg (n = 16); 75.0 kg (n = 25); 62.5 kg (n = 25); 56.25 kg (n = 20); and 50.0 kg (n = 7). This distribution of patients by body weight category was based on the binomial distribution normally found among patients with early-stage HER2-positive breast cancer [Drug costs and healthcare costs to treat 100 patients with either trastuzumab formulation were then calculated assuming the following numbers of patients in each body weight category: 87.5 kg  as the unit of measurement. Hence, costs associated with the total number of vials administered over 18 cycles were calculated, even if some of the last vial’s contents had to be discarded. However, in clinical practice, any drug not used may not necessarily be wasted but rather used for other patients scheduled for treatment in parallel on the same day . This prAll calculations were performed in Microsoft Excel 2016.


These magnetic beads could be utilized to treat contaminated groundwater.Arsenic (As) and lead (Pb) contamination in groundwater is a serious problem in countries that use groundwater as drinking water. In this study, composite beads, called SCM beads, synthesized using stone powder (SP), chitosan (Ch), and maghemite (Mag) with different weight ratios  were prepared, characterized and used as adsorbents for the removal of As and Pb from artificially contaminated water samples. Adsorption isotherm experiments of As and Pb onto the beads were conducted and single-solute adsorption isotherm models such as the Langmuir, Freundlich, Dubinin–Radushkevich (DR), and dual mode (DM) models were fitted to the experimental data to analyze the adsorption characteristics. The maximum adsorption capacities of the SCM beads were 75.7 and 232.8 mmol/kg for As and Pb, respectively, which were 40 and 5.6 times higher than that of SP according to the Langmuir model analyses. However, the DM model had the highest determinant coefficient (R Groundwater contamination has become a serious environmental concern throughout the world. In several countries such as Bangladesh, Vietnam, Pakistan, and China as well as the USA, Hungary, and Mexico, many people are exposed to high levels of arsenic (As) and/or lead (Pb) via the intake of contaminated groundwater ,2,3,4,5.Various technologies for As and Pb removal from contaminated water have been developed, including adsorption, ion exchange, pH control, and precipitation ,8. Adsor3O4 composite material to remove cationic and anionic dyes. Podder and Majumder [2O4 composite to remove As. In addition, many studies have used chitosan as a cross-linker for the synthesis of composite beads, which can bridge and aggregate chitosan, iron-oxide and other powders [Iron oxides have excellent adsorption capacities for As and Pb. Ghosh et al.  reportedMajumder  applied  powders ,33,34. A2) groups, it has a problem with liquidation in which its structure collapses under acidic conditions. A cross-linking agent, such as triphosphate (TPP), can be used to prevent liquidation [3O4 (CCM) composite in which chitosan acts as a cross-linker between the clay and the Fe3O4 and the clay acts as a frame structure. In this study, stone powder (SP) was used to act as a frame structure instead of clay. Untreated SP waste can cause environmental pollution in soil, surface water, and groundwater. Only a small portion of SP is recycled for construction purposes [Chitosan-based adsorbents are interesting because of their eco-friendly properties . Becauseuidation . Cho et uidation  and Ngahuidation  reporteduidation  synthesipurposes  but it iThis study, accordingly, synthesizes a bead containing maghemite, chitosan, and SP for the adsorption of As and Pb and evaluates the performance of the composite bead adsorbent and the factors affecting adsorption, for example, the maghemite/SP weight ratio, bead dose, and temperature. Several adsorption isotherm models, i.e., the Langmuir, Freundlich, Dubnin-Radushkevich (D-R), and dual-mode (DM) models were used to fit the adsorption data.6H11NO4)n with 75–85% degree deacetylation and a viscosity (0.5%) of 5–20 mPa∙s) was purchased from Showa, Japan. Sodium arsenate dibasic heptahydrate  was purchased from Wako, Japan. Lead nitrate (Pb(NO3)2, >99.0%), acetic acid , iron(II) chloride , iron(III) chloride anhydrous , and hydrochloric acid  were purchased from Duksan Co., Ansan, Korea. MES (2-(N-morpholino)ethanesulfonic acid, >98.0%), sodium tripolyphosphate , and sodium bicarbonate  were purchased from Daejung Chem. & Metals Co., Siheung, Korea. Iron(III) nitrate (Fe(NO3)3 9H2O, >98.0%) was purchased from OCI Co., Seoul, Korea.SP was obtained from a masonry mill in Yeongcheon, Korea, air-dried, sieved using a 75 μm mesh (sieve #200), and stored in an airtight plastic bottle before use . Chitosa2O3) was synthesized according to the modification of the Massart method [4OH) was added to a mixture of iron(III) chloride  and iron(II) chloride  to form black-colored settled particles. The particles were separated via centrifugation at 1500 rpm for 10 min, dispersed into a 0.34 M nitrate solution with a pH of 2, and then oxidized to maghemite with iron(III) nitrate at 90 °C for over 1 h. The synthesized maghemite concentration in the ferrofluids was approximately 10 g/L as solids.A ferrofluid composed of maghemite (γ-Fet method . In brieThe synthesis protocol for the SP/chitosan/maghemite (SCM) beads was based on the protocol of Bee et al. . Briefly2 adsorption isotherm. The BET, microscope, and FE-SEM/EDS analyses were conducted at the Instrumental Analysis Center of Kyungpook National University, Korea.The surface shape of the SCM beads was observed using a microscope . A field emission scanning electron microscope  was used to observe the SCM morphology and an energy dispersive X-ray spectroscope  was used to characterize the chemical compositions. Fourier transform infrared spectroscopy (FT-IR) was used for the characterization of the bead structural features. The surface area and pore size of the SCM beads were measured using the Brunauer–Emmett–Teller (BET) method  via the N3 were added. The tubes were capped tightly and shaken at 200 rpm for 24 h in an orbital shaker. After mixing, all tubes were centrifuged at 2000 rpm for 10 min. The supernatant was filtrated through a 0.2 μm membrane filter . The As and Pb concentrations in the aqueous phases were analyzed using an inductively coupled plasma . The pH of the electrolyte solution was in the range of 4–5 adjusted with 0.1 N HCl and 0.1 N NaOH. All experiments were conducted in duplicate.Isothermal adsorption tests were performed to evaluate the adsorption capacity of the SCM beads and SP to As and Pb. All experiments were performed in a 50 mL centrifuge tube . A total of 0.5 g of SCM beads or 1.0 g of SP powder was prepared in screw-cap conical tubes with an available volume of 50 mL, and then metal solutions (As: 0.013–1.33 mmol/L or Pb: 0.965–9.65 mmol/L) with a background electrolyte of 0.01 M NaNOq (mmol/kg), was calculated using Equation (1):C0 is the initial solute concentration (mmol/L), C is the residual solute concentration (mmol/L), V is the sample volume (L), and W is the weight of the adsorbent SCM (×10−3 kg).The solid phase adsorbed amount, 2/g, which was approximately a quarter of that of SP (2.782 m2/g). The pore volume of the beads was in the range of 0.0027–0.0047 cm3/g which was lower than that of SP, whereas the pore size of the beads (20.0–27.9 nm) was similar to that of SP (24.8 nm).The specific area, pore volume, pore size, and pH values of the SCM beads and SP are summarized in Microscopic images and scanning electron microscopic (SEM) images show the shapes of the SCM beads in −1 and a wide crest was observed in the region from 3700 to 3000 cm−1 for all beads. In this region, the absorptions were caused by the N–H and O–H bonds. The bands at 1000 and 780 cm−1 could be attributed to −C−O str and As−O−Fe stretching vibrations, respectively. FT-IR spectra for the bead only, the As adsorbed bead, and the Pb adsorbed bead are shown in q were observed for all adsorption isotherm experiments. The adsorption isotherms of As onto the SCM beads and SP were studied by fitting the experimental data to the representative isotherm models such as the Langmuir, Freundlich, D-R, and DM models, as shown in The experimental results on the adsorption of As and Pb onto the beads and SP are presented here. Based on the data, nonlinear relationships between C and qe) adsorbed onto the beads significantly increased compared with that on SP and increased with the maghemite ratio in the beads (1/1/0.5 > 1/1/0.3 > 1/1/0.1). All adsorptions showed a mixed pattern of C- and L-type isotherms.2), and the squared standard errors (SSE) for As adsorption onto the beads and SP. The R2 values of the As adsorption onto the beads and SP were all higher than 0.90, indicating that all isotherm models can generate a satisfactory fit to the data. Of the models, the Langmuir and DM models fitted the data more accurately than the others.qmL, increased as the maghemite content increased and there was little difference between the qmL values for beads 1/1/0.3 and 1/1/0.5. In the same concentration range, the maximum adsorption capacity increased by 24 times for bead 1/1/0.1 (43.94 mmol/kg), 35 times for bead 1/1/0.3 (65.06 mmol/kg), and 41 times for bead 1/1/0.5 (75.74 mmol/kg) compared with SP (1.84 mmol/kg). However, the b value—adsorption affinity—did not show a consistent pattern.In the Langmuir model, the maximum adsorption capacity, 2 values for the SCM beads and SP were above 0.96 and 0.90, respectively. The KF values were found to be 25, 47, and 44 in the SCM beads 1/1/0.1, 1/1/0.3, and 1/1/0.5, respectively, and the ratio of maghemite was similar at 0.3 or higher. In addition, because the KF value was approximately 10 times higher for the beads than that for SP, it was found that the adsorption affinity of the beads was higher than that of SP. The N values were found to be in the range of 0.66–0.71, indicating nonlinear behavior.In the Freundlich model, the R2 value was higher than 0.96. Because As atoms are 119 pm in diameter and exist in the form of arsenate in water, most of the adsorption appears to have occurred in the pores and surfaces of the beads with pore sizes of approximately 20 nm or more. The qmD values of beads 1/1/0.1, 1/1/0.3, and 1/1/0.5 were 28.07, 47.22, and 44.30 mmol/kg, respectively, and the qmD values of beads 1/1/0.3 and 1/1/0.5 were similar. This value was approximately 25 times higher than the 1.883 mmol/kg measured for SP. Comparing qmD in the D-R model and qmL in the Langmuir model, qmD was smaller than qmL. However, the E values are all less than 8 kJ/mol, indicating that physical adsorption had occurred.2.value of 0.97 or higher for the DM model, which was the same as that for the Langmuir model. The qmDM value of the DM model and the qmL value of the Langmuir model were very similar, and the KpDM value was very low (less than 0.049), indicating that the monolayer adsorption was dominant.In this study, the dual mode (DM) model, which is a model combining the Langmuir model and the linear model, was also applied to analyze whether monolayer or multi-layer adsorption occurred. qe) adsorbed onto the beads significantly increased compared with that adsorbed onto SP and increased with the maghemite ratio in the beads (1/1/0.5 > 1/1/0.3 > 1/1/0.1). 2 values for all model fitting results were greater than 0.618 in bead 1/1/0.3. The R2 value of the DM model was the highest of all the models.qmL) was 222.2, 200.8 and 232.8 mmol/kg for beads 1/1/0.1, 1/1/0.3, and 1/1/0.5, respectively, showing similar results regardless of the ratio. Comparing the qmL values with that of SPs (41.8 mmol/kg), those of bead 1/1/0.1 increased by 5.3 times, bead 1/1/0.3 increased by 4.8 times, and bead 1/1/0.5 increased by 5.6 times. The adsorption affinity, b, also increased with the maghemite content. The R2 for the Langmuir model was in the range of 0.797–0.909. This makes it difficult to conclude whether the adsorption of Pb was a monolayer adsorption.In the Langmuir model, the maximum adsorption capacity  of the models. This is because the beads have various adsorption sites composed of a composite component with a difference in adsorption affinity between the maghemite and SP. Unlike As adsorption, the KpDM values in the model for Pb adsorption were high at 15.7, 14.9, and 9.1 for beads 1/1/0.1, 1/1/0.3, and 1/1/0.5, respectively, showing a decrease with increasing maghemite content. Conversely, the KpDM value for the Pb adsorption onto SP was nearly 0. In addition, the qmDM value was 66.0, 96.9, and 190.7 mmol/kg for beads 1/1/0.1, 1/1/0.3 and 1/1/0.5, respectively, increasing in proportion to the maghemite content.The DM model can analyze the dominant model between the physical multi-layer adsorptions and the monolayer adsorption. Its RF value in the model is related to the adsorption affinity. As shown in F values increased with increasing pH, with 102.6 (at pH 7.0) > 100.3 (at pH 5.5) > 53.3 (at pH 4.0) for As adsorption and 512.7 (at pH 7.0) > 440.7 (at pH 5.5) > 394.4 (at pH 4.0) for Pb adsorption, indicating that the Pb adsorption affinity was higher overall than the As adsorption affinity.pK in the linear model was used as a thermodynamic parameter related to the Gibb’s free energy change, ΔG0 (kJ/mol) during adsorption. The changes in the enthalpy, ΔH0 (kJ/mol), and the entropy, ΔS0 (J/mol/K), were also calculated using the following equations:R is the gas constant (8.314 J/K/mol), T is the absolute temperature in Kelvin, and pK is the partitional coefficient.A thermodynamics analysis was conducted for the adsorption of As and Pb onto the beads to estimate whether the reaction occurred spontaneously. The partition coefficient p) values for As and Pb adsorption increased as the temperature increased from 293 to 313 K. As shown in The effect of temperature on the adsorption of As and Pb onto the SCM beads is shown in SCM composite beads were prepared with different mass ratios of 1/1/0.1, 1/1/0.3, and 1/1/0.5 of SP/chitosan/maghemite, respectively, for the adsorption of As and Pb. The composite beads had maximum uptakes of 1.83 and 50.0 mmol/kg for As and Pb, respectively. The optimum mass ratio of SP, chitosan, and maghemite in the bead for the maximum adsorption capacity was 1/1/0.5. The experimental data were analyzed using several adsorption isotherm models. From the results of the model fitting, the beads had multiple adsorption sites with different affinities resulting from the inclusion of maghemite compared with SP and the DM model was the best model to describe these differences. The adsorption also depended on the pH and temperature. The thermodynamic study indicated that the adsorption reactions of As and Pb onto the beads are endothermic and spontaneous resulting from the large increase in the entropy change. In conclusion, beads could be used as good effective adsorbents for As and Pb removal from water resources, including rivers and groundwater.


Solemoviridae. This virus had less than 90% amino acid sequence identity in the products of both ORF0 and ORF1. According to the polerovirus species demarcation criteria set by the International Committee on Taxonomy of Viruses, this virus should be assigned to a new polerovirus species, for which we propose the name “cotton leaf roll virus”.Cotton is the most important fiber crop worldwide. To determine the presence of viruses in cotton plants showing leaf roll and vein yellowing symptoms in Henan Province of China, a small RNA-based deep sequencing approach was performed. Analysis of the de novo-assembled contigs followed by reverse transcription PCR allowed the reconstruction of watermelon mosaic virus and an unknown virus. The genome of the unknown virus was determined to be 5870 nucleotides in length, and has a genomic organization with characteristic features of previously reported poleroviruses. Sequence analysis revealed that the virus was closely related to, but significantly different from, cotton leafroll dwarf virus, a polerovirus of the family  Plant viruses have caused significant yield and quality losses to a wide range of economically important crops . As obliPotyviridae is the largest family of plant RNA viruses that are ascribed to substantial yield losses to agricultural, pastoral, horticultural, and ornamental crops globally [Potyviridae member viruses. Viruses belonging to the genus Potyvirus constitute the largest group of the family Potyviridae and are transmitted by aphids. The typical genome of potyviruses has a single-stranded, linear, positive RNA that is made up of approximately 10,000 nucleotides. It encodes a long open reading frame (ORF) that is processed into functional proteins by three self-encoded proteases. An additional small ORF, PIPO, is formed from RNA polymerase slippage during viral genome replication [Potyvirus genus and viruses from other genera. Although the outcomes of coinfection with two individual viruses may vary, coinfection of a potyvirus with another distinct virus often results in an enhancement of symptom severity and yield loss [The globally . Twelve lication . Besideseld loss . For exaeld loss ,10.Solemoviridae is another important family of plant linear (+) ssRNA viruses that are divided into four genera   [Polerovirus genus are transmitted by aphids and possess a genome of about 5300 to 6000 nucleotides in length. The typical genome of a polerovirus encodes seven ORFs  [The st 2021) . VirusesF3-ORF5) . The ORFF3-ORF5) .Polerovirus were identified from the diseased cotton plants through Illumina-based deep sequencing.Cotton is the most important fiber crop worldwide. It is also an excellent multipurpose crop providing a source of feed, foodstuff, biofuel, and oil production. Previous studies have shown that, in some of the major cotton-producing countries, cotton crops are often severely affected by diseases caused by infection with cotton leaf curl begomoviruses or and cotton leafroll dwarf virus ,14,15. ICotton plants showing leaf roll and vein yellowing symptoms were collected from Anyang City, in the Henan province of China, in May 2016 . Leaf tiTM III reverse transcriptase . The first-strand cDNA was then PCR amplified and subjected to Illumina Hiseq2000 sequencing . The raw reads were filtered by trimming adapter sequences and removal of poly A/T/C/G reads and low-quality reads. The clean sRNA reads ranging from 18 to 26 nts in length were mapped to the cotton genome, and the clean unmapped sRNA reads were assembled using the Velvet program [http://www.ncbi.nlm.nih.gov/, accessed on 15 January 2017).Small RNA library was constructed as described . In brie program . AssemblThe presence of candidate viruses in diseased leaf tissues was validated by reverse transcription PCR (RT-PCR). Primers were designed according to the sequences of assembled contigs of virus-derived small interfering RNAs (vsiRNAs).TM II First-Strand Synthesis System (Invitrogen) with a random hexamer and virus-specific primers . For cloning of the full-length viral genomes, first-strand cDNA synthesis was performed using the SuperScript primers . AmplifiFor detection of the negative strand of watermelon mosaic virus, reverse transcription was performed using a tagged primer WMV-NS-RT , and verified by comparison with previously annotated ORFs from CLRDV and WMV reference sequences deposited in GenBank.Sequences were processed using the Lasergene 7 SoftWare . Open reading frames (ORFs) were predicted using the ORF Finder program .Quantitiave RT-PCR (qRT-PCR) was performed using a TransStart Green qPCR Supermix  and the Roche LightCycler 96 system  following the manufacturer’s recommendations. The relative expression of viral RNA was determined by normalizing to the expression of the internal control Potyvirus, family Potyviridae), and 28 contigs covering 3326 nts were annotated to have a high similarity to the cotton leafroll dwarf virus (CLRDV) .To identify virus(es) present in the diseased cotton plants , leaf tiTo validate and obtain the full-length viral genome sequences, several RT-PCR amplifications were carried out using primers  designedPotyvirus, the Henan isolate of WMV encoded a single polyprotein (133–9780 nt) which was predicted to contain 11 protein products, including 10 mature products proteolytically processed by viral proteases and an 11th protein P3N-PIPO embedded in P3 by +2 ribosomal frameshifting .Similar to members in the genus shifting A. A phylshifting A. Furtheshifting B. AccordSimilar to previously reported poleroviruses, the genomic organization of the Henan isolate comprised 5′ block and 3′ block that are separated by a 79-nt noncoding sequences. The 5′ block consisted of three predicted ORFs: ORF0, ORF1, and ORF1-ORF2. The 3′ block contained four ORFs, ORF3, ORF4, ORF3-ORF5, and an additional short non-AUG initiated ORF, called ORF3a, that is positioned upstream of ORF3 B. The 5′The closest relatives of the 5870-nt viral sequences are the nine CLRDV isolates previously identified from Argentina, Brazil, and USA ,21,22,23Solemoviridae. As shown in Polerovirus and was most closely related to the nine CLRDV isolates. A closer view of the phylogenetic tree showed that the Henan isolate was in a distinct subclade from the nine CLRDV isolates  is proposed (GenBank Accession Number: OK050525).To better understand the relationship of the Henan isolate to other poleroviruses, a phylogenetic tree was constructed based on the full-length genomic sequences of the Henan isolate and members of the family isolates . Phylogeisolates . CurrentTo determine the abundance of WMV and CLRV in the collected cotton plants, qRT-PCR was used to analyze the relative expression of WMV and CLRV RNA. The results showed that the RNA accumulation level of CLRV was about 22 fold to that of WMV , suggestPolerovirus genus.The cutting-edge deep sequencing technology and bioinformatics have been proven to be a reliable and efficient approach for virus identification, which does not rely on prior knowledge or sequence of the virus of interest. Therefore, the development of this technology has boosted the discovery of novel viruses. In this study, we identified and characterized coinfection of cotton plants with a watermelon mosaic virus and a novel member of the Solemoviridae also revealed that the identified virus grouped with poleroviruses, but separated from members of the Enamovirus, Polemovirus, and Sobemovirus genera. A detailed BLASTn and phylogenetic analysis showed that this virus was closely related to CLRDV, a polerovirus that has been described to cause cotton blue disease in Argentina, Africa, Asia, Brazil, and the United States [Polerovirus genus utilizes a >10% amino acid sequence difference in any of the viral products as a new species threshold [The polerovirus identified in this study shares a typical genome structure with known poleroviruses. Phylogenetic analysis of the complete genome sequences to reported typical species of the family d States ,23,24,25hreshold . In the hreshold . SequencPotyviridae, genus Potyvirus, PVY) and potato leafroll virus  in potato cropping systems, are common in nature [Potyvirus has been found to co-infect with a polerovirus in cotton plants. The biological and epidemiological consequences of mixed viral infection are unpredictable and largely depend on the combinations of virus–virus and virus–host interactions, which are usually categorized as synergistic and antagonistic interactions [Nicotiana clevelandii by PLRV and PVY increases PLRV accumulation and helps PLRV to invade non-phloem tissue [Coinfections of the same host plant with heterologous viruses, such as potato virus Y (family n nature . Howeverractions . Synergiractions . While pm tissue . An intem tissue . Since b


This system differs from other plant–pathogen interaction systems, in which plant R genes recognize a single type of gene or gene family because almost all virus genes with distinct structures and functions can also interact with R genes as Avr determinants. Thus, research conducted on viral Avr-R systems can provide a novel understanding of Avr and R gene product interactions and identify mechanisms that enable rapid co-evolution of plants and phytopathogens. In this review, we intend to provide a brief overview of virus-encoded proteins and their roles in triggering plant resistance, and we also summarize current progress in understanding plant resistance against virus Avr genes. Moreover, we present applications of Avr gene-mediated phenotyping in R gene identification and screening of segregating populations during breeding processes.In the plant immune system, according to the ‘gene-for-gene’ model, a resistance  Plant viruses contain single-stranded or double-stranded RNA or DNA genomes and vary substantially in their genome structure and organization. Moreover, limited viral genome sizes and coding capacities have resulted in evolution of multifunctional proteins that are involved in different steps in the virus life cycle, including replication, movement, encapsidation and transmission. On the other hand, as obligate intracellular parasites, plant viruses absolutely depend on the host cell machinery to multiply, move throughout the plant and spread to susceptible hosts. During infection, viruses consume a substantial amount of host resources; subsequently, disease symptoms develop as a consequence of disruptions of the cellular machinery required for plant physiology and natural growth, and these disruptions eventually result in developmental abnormalities and other phenotypic manifestations. Viruses can be critical players in pathogenesis through direct or indirect interactions. However, in some plant species or varieties, virus-encoded proteins can sometimes act as determinants in plant defense responses and as host-controlled pawns to elicit extreme resistance (ER).According to the zigzag model of plant–pathogen interactions, the plant innate immune system is broadly divided into two different layers: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) . PTI is R genes were identified in 1984 [On the other hand, to counteract ETS, plants have evolved intracellular resistance (R) proteins that directly or indirectly recognize pathogen effectors or Avr factors to activate ETI and trigger the second layer of defense. ETI is often manifested as a hypersensitive response (HR), characterized by rapid cell death, production of reactive oxygen species (ROS) and salicylic acid (SA) induction and expression of defense-related genes ,20,21. S in 1984  and 1994 in 1984 , increasThe CP, also known as the capsid protein, encapsidates and protects viral genomes from damage. Early expressed CPs function in disassembly of parental virions and have roles in assembly of progeny virions during the final infection steps. However, more and more evidence has shown that CPs of all plant viruses are multifunctional and have various roles during different replication stages, ranging from early to late events in the infection cycle. The diversity of these functions in different viral systems includes virus transmission by specific vectors, translation of viral RNA, regulation of intercellular and systemic movement of the virus, suppression of both post transcription gene silencing (PTGS) and transcription gene silencing (TGS), as well as determination of symptomatology and pathogenesis ,25. OwinR gene-mediated host defenses has been extensively characterized. The CPs of Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV), Bell pepper mottle virus (BPeMV), Paprika mild mottle virus (PaMMV), Obuda pepper virus (ObPV), Pepper mild mottle virus (PMMoV), Potato virus X (PVX) and Mungbean yellow mosaic virus (MYMV) each serve as Avr factors that elicit resistance controlled by cognate dominant R genes  and resistance-breaking (RB) strains [N′-mediated recognition requires maintenance of the CP three-dimensional structure, either directly, or through specific structural motifs [N′ gene and its orthologues were recently cloned from N. sylvestris and other TMV resistant Nicotiana species and shown to encode CC-NB-LRR type proteins [Nicotiana section Alatae species [Nicotiana section Alatae carry functional N′ orthologues [N′ and N′ orthologues might have been inherited from a common ancestor followed by evolution to confer tobamovirus and tombusvirus resistance to Nicotiana genus species. In pepper, a broadening spectrum of resistance to seven known pepper-infecting species of tobamoviruses  is conferred by the corresponding localization (L) alleles [L gene alleles also encode CC-NB-LRR type resistance proteins with the ability to elicit resistance responses to different tobamoviral CP Avr effectors [N′ and its Nicotiana orthologues and the L alleles from pepper mediate resistance against tobamoviruses by recognizing the CP [Solanum ancestor. However, a resistance gene evolution assay indicates that the L gene from pepper is not an N′ orthologue, suggesting that tobamovirus resistances in pepper and Nicotiana originated independently [R genes within the same locus recognize different CP proteins from overlapping virus species indicating that the conserved R proteins are able to recognize similar structures but with an adapted spectrum. These results also support the idea that interactions between L genes or N′ orthologues and tobamovirus CPs serve as good systems for study the mechanisms and evolution of virus perception by plants.TMV CP was identified as an Avr responsible for eliciting host ER responses during interactions with the  strains ,29,30. Sl motifs ,32. The proteins ,34. Inte species . Moreove alleles . L gene ffectors ,38. Sincg the CP ,34, it sRx1 gene, which encodes a class of CC-NB-LRR R proteins in potato [Rx1 in an auto-inhibited (off) state [RX1 does trigger an HR upon overexpression of the Avr PVX CP or under high PVX concentrations [Rx1 leads to translational arrest of PVX CP transcripts and compromises extreme resistance against PVX [Nx, also confers resistance to the PVX CP. However, ER by the Rx1 gene is induced via interactions with PVX CP conserved amino acids (aa) 121–127, whereas the Nx gene confers resistance to PVX through the recognition of PVX CP aa 62–78 [Another representative study involves PVX CP-elicited ER mediated by the n potato . Under vf) state ,80. Upontrations . An additrations . Howeverinst PVX . Thus, maa 62–78 ,42,83.Arabidopsis thaliana ecotype Di-17, Turnip crinkle virus (TCV) CP P38 functions as the Avr resistance determinant for the HRT CC-NB-LRR R protein [N terminus has been shown to be involved in eliciting resistance responses [N terminal nuclear localization domain is important for elicitation of host resistance responses and could be a key trigger for HRT-dependent resistance to TCV [A. thaliana ecotype C24, RCY1, which encodes a CC-NB-LRR class R protein, was isolated and identified as the first R gene conferring resistance to Cucumber mosaic virus (CMV) [RCY1 Avr resistance response also mapped to the CP [RCY1 and HRT R genes belong to HRT/RPP8 gene family, and the RCY1 locus in ecotype C24 were was found to be allelic to HRT in Dijon-17. These functionally divergence genes therefore seem to have evolved via recombination of ancestral genes [RCY1 and HRT genes [RCY1 and HRT are elicited by completely different ligands. Another possibility is that the single CPs of CMV and TCV, or their possible complexes with other host components  may have highly similar CP protein folding domains [In the  protein ,84. By uesponses . Moreovee to TCV . In anotus (CMV) . The CMVo the CP . Interesal genes . More inRT genes ,84. Ther domains .R gene-driven effector triggered immunity (ETI) causing HR  that has been widely employed in tomato breeding programs that have released numerous commercial cultivars. Recently, Ty-2 was cloned and shown to encode a CC-NB-LRR protein [Ty-2 along with the Rep/C1 and C4 genes of the RI and RB TYLCV strains, respectively, in N. benthamiana, only the RI TYLCV strain Rep/C1 protein consistently elicited a HR when co-expressed with the Ty-2 protein. This result clearly indicates that the Rep/C1 protein is the Avr determinant triggering the Ty-2 based (strain-specific) resistance response [ protein . Using aresponse .N gene was cloned almost twenty years ago and shown to be a member of the TIR-NBS-LRR class of plant disease resistance genes [N gene. Beachy’s group initially reported a role for the 126/183-kDa replicase, but not the CP or MP, in induction of HR in tobacco containing the N gene [N-mediated defense responses [N-mediated ER [As the first and one of the best studied plant virus resistance genes, the ce genes . Subseque N gene , Later, esponses . Ultimatiated ER . They aliated ER . The amiPvr4 gene encodes a broad-spectrum CC-NBS-LRR type resistance protein known to elicit resistance against multiple potyviruses, including Pepper mottle virus (PepMoV), Pepper severe mosaic virus (PepSMV) and Potato virus Y (PVY) in Capsicum annuum [Pvr4 resistance in pepper plants [Avr determinant for two resistance genes, TuRB01 and TuRB05, in the AA subgenome of Brassica napus [The m annuum . Kim et r plants . The cylca napus ,56.Virus cell-to-cell and long distance movement throughout the plant from initial infection sites are controlled by specific viral MPs. Generally, plant virus movement is divided into three steps: (I) intracellular movement in which virus is trafficked along different organelles within a single cell from the sites of replication to the plasmodesmata ; (II) inR gene products. One of the best-characterized systems is the interaction between the Tomato spotted wilt virus (TSWV) NSm movement protein with the tomato Sw-5b and tobacco RTSW resistance gene proteins. Sw-5b belongs to the CC-NB-LRR type immune receptors and contains an extended N-terminal Solanaceae domain (SD) [21), which is highly conserved among the American-type orthotospoviruses, but not the Euro-Asian-types, is sufficient to trigger Sw-5b-mediated HR [21, the SD domain functions as an extra sensor to detect low levels of NSm or NSm21 and enhance resistance of Sw-5b [RTSW, another R locus from wild tobacco N. alata that confers ER to TSWV, has been introgressed into cultivated tobacco. By using two different transient expression systems, we showed that the NSm protein of TSWV acts as an Avr determinant of RTSW-based resistance [RTSW and Sw-5b behave as single dominant genes that confer ER by interacting with the same NSm protein encoded by the same virus, they recognize different elicitor domains (EADs) [Tm-2 and (2)Tm-2 genes. The 30-KDa MP of ToMV elicits both Tm-2 and (2)Tm-2 resistance, but has different interacting domains [Several examples have shown that viral MPs can elicit an HR response via interactions with ain (SD) ,58. Thisain (SD) ,97,98. Tain (SD) ,60. Subsiated HR . In addiof Sw-5b ,101. RTSsistance . Moreovesistance ,102. Mors (EADs) . The sam domains  because  domains . Brachypodium distachyon Bd3-1 inbred line harbors a resistance gene designated Bsr1 that interacts with the Barley stripe mosaic virus (BSMV) ND18 triple gene block 1 (TGB1) movement protein to elicit ER during infection. Lee et al. have shown that two amino acids within the protein are required to elicit resistance, and that the BSMV Norwich strain has a two amino acid changes in TGB1 that abrogates necrosis to enable systemic infections [Nb-Mediated ER, and that an isoleucine residue at position 6 of the MP protein is required for activation of the Nb response [The model grass fections . In potaresponse . More inresponse . TherefoR genes to elicit ETI, which acts as a second plant defense layer [RNA silencing  functions in endogenous gene regulatory and exogenous antiviral mechanisms in plants . As a cose layer . Avr determinant for Tsw-based resistance in pepper [Tombusviridae also support the conclusion that SSR and HR elicitation involve independent motifs. Among the four clades in the phylogenetic tree of 54 CPs tombusvirids, two separate CP clades triggered an HR in Nicotiana species of section Alatae but did not have silencing suppressor activity, whereas in one clade the CP functioned as an SSR, but lost the capacity to trigger HR in Nicotiana species [The orthotospoviral NSs silencing suppressor protein is a well characterized RSS that conducts local and systemic silencing suppression functions by binding small and long dsRNAs ,115. Recn pepper . Considen pepper . Recent  species .N. glutinosa RPO1 gene elicits ER against poleroviruses Turnip yellows virus (TuYV) and Potato leafroll virus (PLRV), and is inherited as a single, dominant resistance allele. The P0 proteins from TuYV, PLRV and Cucurbit aphid-borne yellows virus (CABYV) were found to elicit an HR in N. glutinosa accessions containing the RPO1 locus. To dissect important roles of the P0 F-box motif in autophagic degradation of AGO1 proteins, motif mutants were constructed to test requirements for elicitation of RPO1-mediated HR. The results showed that P0 mutants with substitutions in the F-box motif that abolished SSR activity were unable to elicit HR, suggesting that HR induction requires a functional P0 protein [Gossypium hirsutum), Cbd is a single dominant gene conferring ER to the polerovirus, cotton leafroll dwarf virus (CLDV). Agrofoglio and coworkers characterized the SSR activity of the P0 proteins encoded by wildtype (WT) CLRDV and a RB CLRDV isolate and evaluated the roles of these proteins in breaking Cbd-resistance in cotton plants. They found that WT and RB CLRDV P0s behave similarly during RNA silencing interference; however, RB CLRDV P0 enabled WT CLRDV and a chimeric infectious clone to systemically infect Cbd-resistant cotton varieties. Therefore, the CLRDV P0 protein is also an Avr determinant that functions in Cbd-based resistance [The Polerovirus P0 protein is another well-known SSR that can mediate degradation of ARGONAUTE1 (AGO1), one of the most important components in RNA silencing and antiviral responses . The N.  protein . In cottsistance .avr gene, P6 of CaMV was identified through its capacity to elicit an HR in Datura stramonium and N. edwardsonii, by dissecting a series of gene swaps between infectious clones of different strains of CaMV [N. glutinosa, but segregated independently from the cell death responses of N. edwardsonii or N. clevelandii [R genes affecting these processes have not been isolated. P6 has also been recognized as a versatile viral protein that has key roles in several steps of virus infections [In contrast to a simple classification in which ER caused by viral elicitors falls into the four categories above, research within the past few years shows that ER determinants are largely spread throughout the viral genome ,119. Add of CaMV ,72. Subsvelandii ,121, altfections . For exafections ,123,124.fections ,127,128.fections ,130.sto resistance, which confers ER against PVY and related viruses from the wild relative S. stoloniferum, has been widely employed in potato breeding programs as an importance resistance trait. Recently, the Rysto gene was cloned and found to encode a TIR-NB-LRR protein [Avr determinant for Rysto was determined more than twenty years ago, when Mestre et al. demonstrated that the NIa proteinase (NIaPro) from PVY is an elicitor of Rysto-mediated resistance [sto resistant but not in rysto susceptible potato [stoRy-mediated potyviral resistance [sto gene was isolated because similar transient expression strategies with different viral proteins from PVY and Potato virus A (PVA) indicated that Rysto recognizes the CP but not other proteins as Avr factors in Rysto transgenic N. tabacum plants [Ry protein . Neverthsistance . In thise potato . In a susistance . Howeverm plants . TherefoGlycine max), the Rsv1 gene confers resistance to the N strain of soybean mosaic virus (SMV), one of the most devastating potyviruses that causes huge economic losses in soybean production worldwide. Through construction of a series of chimeras and site-directed mutants, Eggenberger and colleagues mapped SMV P3 and helper-component proteinase (HC-Pro) components involved in elicitation of Rsv1-mediated ER. Since P3 and HC-Pro are cistrons within a read-through protein, it seems that HC-Pro and P3 are recognized by Rsv1-mediated defense responses as a precursor polyprotein [Rsv1 locus may be a reason why the recognition sites are distributed throughout the HC-Pro and P3 cistrons. Multiple distinct resistance genes, likely belonging to the NB-LRR class, have been discovered within the Rsv1 locus [Avr determinant for two dominant resistance genes, TuRB03 and TuRB04, in the brassica-TuMV interaction system [In soybean plants  and RP28 genes can be easily and quickly isolated from a 200 Rx homologue library from potato and a 99 candidate R gene library from pepper by using Agrobacterium coinfiltration approaches [R genes encoding NB-LRR type receptors are frequently members of large gene families, organized in complex clusters of paralogous genes in the plant genome. Of course, several additional candidate R homologues still may need to be cloned and identified even if located within a very narrow chromosome fine mapping region.In early studies, viral Avr factor identification was conducted via several approaches including gene swaps between resistance and susceptible virus strains or mutants, and ectopic expression of Avr candidate genes within a virus vector. Subsequently, agroinfiltration has been shown to be a more convenient and powerful tool for screening of viral proteins capable of triggering HR in R-containing plant hosts. According to the ‘gene-for-gene’ theory, one approach ,132. In proaches ,132. TheR gene families. The high sequence similarity of encoding and flanking regions of R gene alleles by marker assisted genotyping are huge challenges. However, coevolution and diversified selection of R genes and the pathogen’s Avr effectors often result in multiple recognition regions between a single R gene with each allele and corresponding species or strain-specific Avrs. Several examples of allelic series of virus R genes are known in plants; for example the L1, L2, L3, and L4 alleles confer broad spectrum of resistance to different tobamoviruses by recognizing their CP elicitors [Avr gene-based phenotyping has been established and applied to rice blast disease resistance identification in the field by using different Avr factors [Identification of new candidates for virus resistance is a prerequisite for effective utilization of diverse germplasm in breeding programs. Multiple alleles arising by rapid evolution through tandem and segmental gene duplications, recombination, unequal crossing-over or point mutations are common within licitors . Therefo factors .Avr gene-based diagnostic approaches can provide rapid disease resistance tests that overcome limitations of virus sap inoculations and greatly improve the efficiency and accuracy of test plant inoculations [For fine mapping and cloning of virus resistance genes, a precise disease resistance test is a prime requirement for resistance identification in segregation populations, e.g., F2, BC, RIL or NIL populations. Traditional virus disease resistance tests performed in mapping or breeding programs involves virus infectious clones, insect vectors, or sap mediated virus inoculations. However, the obvious shortcomings of these methods, including low accuracy, complicated operations, time consuming experiments, and labor intensive trials has limited their wide application. However, our experience has indicated that ulations .Avr gene identification is relatively easy due to small virus genome sizes and limited numbers of gene products. Most virus Avr genes have been matched with an R-gene type NB-LRR receptor , viral receptor . Howeveryviruses , Y-1 in e to PVY , Pv1 andot virus , Ctv resza virus  and BcTu to TuMV . Thus, f21) and Sw-5b that revealed a novel two-step recognition mechanism involving the SD and LRR domains of Sw-5b [N gene and TMV p50 mediated by an intermediate protein NRIP1 [(2) involving an intermediate NbMIP1 [(2), other Avr and R proteins interactions were limitedly characterized experimentally. Thus, a major challenge for the future is to identify the precise interaction mechanisms between viral Avr factors and their matching NB-LRR type receptors, as it is possible such multi host interactions are common. The resolution of such mechanisms may require more powerful technological tools to detect and analyze subtle signaling complexes. Nevertheless, several novel experimental approaches, including TurboID-based proximity labeling, high throughput omics analyses, protein–protein interaction networks and machine learning technologies have recently demonstrated their potential for comprehensive understanding of complex biological process such as Avr/R protein signaling cascades [Previous studies with virus Avr-R interactions have provided new insights and paved the way to plant innate immunity studies of other pathogens. Among these are investigations of direct interactions between NSm  functions on the plasma membrane [+ hydrolysis that can trigger plant immunity responses. Future steps will require advances in structural studies, understanding patterns and mechanisms of R protein recognition and activation of viral Avr factors. Addressing such fundamental mechanisms will clarify major gaps in our understanding of plant virus defense networks.Notably, recent cryo-EM structural and functional analyses of CC-NB-LRR (ZAR1) and TIR-NB-LRR (ROQ1 and RPP1) proteins have shed new light on resistosome signaling mechanisms and provide an excellent template for other CC- and TIR-NB-LRR protein functional cascades ,146,147.membrane  and Sw-5membrane ,148. It


In order to explore the fractal characteristics of particle size distribution (PSD) of various minerals in loess and lime-treated loess, the Q4 undisturbed loess and lime-treated loess were studied. From the perspective of multi-scaled microstructure, the internal characteristics of loess were observed and the regularity statistics were carried out from a macroscopic view. Fractal theory was used to quantitatively study the distribution of mineral particles in undisturbed loess and lime-treated loess. It was found that the skeleton particles of undisturbed loess were obvious and the structure of soil was loose. While that of lime-treated loess decreased, the fine particles were connected with each other, and the structure of soil changed from loose to dense. The three mineral particles in the undisturbed loess and lime-treated loess did not accord with the single fractal distribution characteristics, but the total particles had fractal characteristics. The percentage content of the mineral particles in the soil varied greatly with the particle size. In addition, the non-uniform degrees of mineral particles in the two soils from large to small were carbonate minerals of lime-treated loess, carbonate minerals of undisturbed loess, quartz minerals of lime-treated loess, feldspar mineral of lime-treated loess, feldspar mineral of the undisturbed loess, and the quartz mineral of the undisturbed loess. This paper provided a basis for the future study of the different soil mechanical properties of undisturbed loess and lime-treated loess. Loess is a widely-distributed typical structural soil ,2,3. TheIn recent years, important achievements have been made in the study of soil structure by using fractal and multifractal theory, from the micro scale (μm) to the macro scale (km) ,6. The e4 undisturbed loess and lime-treated loess were quantitatively analyzed, and the distribution characteristics of single fractal and multi-fractal of three mineral particles in the two soils were discussed, thus providing a basis for studying different soil mechanical properties of natural loess and lime-treated loess.However, due to the limitation of test technology, most of these studies only analyzed the fractal characteristics of the overall particle size distribution of soil, while fewer studies focused on the fractal characteristics of different mineral particles under different soils. The SEM-EDX study was widely used in cement and concrete research ,18,19. I4 undisturbed loess sample was taken from the exploratory well of Xining Qinghai-Tibet Science and Technology Museum, which is located in Xining City, Qinghai Province. The untreated loess sample, 6~8 mm in height, was cut from the original ring knife sample and was pressed into the soil extractor. The main physical properties are as follows: natural water content w is 16.7%, soil specific gravity Gs is 2.70, natural bulk density r is 16.3 kN/m3, void ratio e is 0.93, dry density ρd is 1.4 g/cm3, liquid limit is 24.8%, and the plastic limit is 15.4%. A part of the soil samples was crushed, air-dried, and sieved through an aperture of 2 mm. Lime was weighed, and according to a mass ratio of 100:7, mixed evenly with loess to prepare the lime-treated soil samples with water content of 16.7% and dry density of 1.39 g/cm3. It was pressed into the soil extractor for sample preparation, drying, curing, coarse grinding, fine grinding, polishing until the surface of the soil sample showed flatness and smoothness, which can be observed by electron microscope scanning, and then it was coated with gold sprayer for further use.The QThe sample was scanned with a JSM-6390A scanning electron microscope at a magnification of 500 times. After scanning, an energy spectrum base map and a surface scanning image of seven elements Si, Al, Ca, K, Fe, Mg, and Na were obtained; 320 images (including 40 base map and 280 EDS photo for 7 different elements) were obtained for each sample . CorrespIn order to ensure complete and seamless splicing, the images were spliced in the form of an S-shaped route with a horizontal overlapping width of 1/4 and a vertical overlapping length of 1/3. The multi-scale microscopic image of mineral composition distribution in the two soils was obtained and transformed from micron to millimeter scale, as is shown in After the microscopic image was processed, the chromatic value  of unknown minerals was obtained by averaging. According to the formula ΔE = ((Δa)2 + (Δb)2)1/2, the difference between the chromaticity value of unknown minerals and the standard chromaticity value of minerals was calculated. Δa = a − a1, Δb = b − b1, where  is the standard color of mineral A. If ΔE < 1, the mineral was identified as A. The identification results of mineral image recognition were compared with those of energy spectrum to verify the accuracy of the method. The validation schematic is shown in Based on this method, quartz, feldspar, and carbonate mineral particles in untreated loess and lime-treated loess were identified respectively, and the particle size parameters of each mineral particle in the two soils were extracted.It can be observed from The particle size parameters of each mineral particle in natural loess and lime-treated loess were extracted, and the particle size interval I =  was selected according to the logarithmic equidistant method. The distribution of different mineral particle sizes was calculated, and the spatial distribution characteristics were analyzed and compared. The particle size distribution curves of various minerals in the two soils are shown in It can be seen from From the whole mineral particle size distribution curves, compared with undisturbed loess, the particle size of all mineral particles in lime-treated loess decreased. This is consistent with the decrease of mineral particles in lime-treated loess observed directly above.Soil is a complex porous medium with fractal characteristics, and the fractal can be defined by the relationship between particle size and number of soil particles .(1)N .(3)ui .(4)Dq=lWhen Singularity index of mineral particle size distribution tion (5) .(5)α(q)The multifractal spectrum function In the range of It can be speculated that in the process of crushing and compaction, large particles are crushed to many small particles and a series of chemical and physicochemical reactions occur between lime and soil to give rise to cements, such as calcium carbonate and crystal calcium hydroxide, etc. The aggregation of clay colloid particles makes the soil structure dense.From the whole mineral particle size distribution curves, compared with undisturbed loess, the particle size of all mineral particles in lime-treated loess decreases. This is consistent with the decrease of mineral particles in lime-treated loess observed directly above. In terms of the non-uniformity of mineral particle distribution, the carbonate mineral particle distribution in lime-treated loess is the most uneven. This is the result of a series of chemical and physicochemical reactions in the soil after lime is added, which gives rise to cements, such as calcium carbonate and crystal calcium hydroxide.According to Equation (2) and the PSD curves of each mineral particle in natural loess and lime-treated loess, For different properties of soil, the fractal dimension of particle size distribution reflects the particle size and distribution uniformity. The larger the fractal dimension is, the smaller the particle size of soil mineral particles is, the higher the fine particle content is, and the more uneven the texture is. The fractal dimension in equation 2 reflects the characteristics of particle size distribution of soil mineral particles, and has a clear physical meaning. It shows that when than 3.0 . Since tIt can be seen from It can be seen from the particle size distribution curve of mineral particles in According to the multifractal theory, when As is shown in On the basis of the multifractal analysis of three kinds of mineral particles in undisturbed loess and lime-treated loess, the generalized dimension spectrum curve For non-uniform fractal, mity was . PSDs ofThe multifractal singular spectrum of PSD of three different minerals in undisturbed loess and lime-treated loess is shown in Symmetry eft hook . From FiAccording to the multifractal theory, tructure .0, remaining unchanged with the increase of q. The larger Δα is, the more uniform PSD is. Its distribution characteristics can be described by multifractal rather than single fractal. Takele et al.  believedThrough image scanning, processing, and mineral composition identification, the obtained multi-scale microstructure images were more representative and could better present the characteristics of macroscopic soil samples. In this work, fractal theory was used to quantitatively study the distribution of mineral particles in undisturbed loess and lime-treated loess. The following main conclusions may be drawn:The skeleton particles of undisturbed loess were obvious and the soil structure was loose, whereas the skeleton particles of lime-treated loess decreased, fine particles were connected with each other, and the soil structure changed from loose to dense. The particle size of each mineral particle in lime-treated loess decreased, and the distribution of carbonate mineral particles was the most non-uniform.Mineral particles in undisturbed loess and lime-treated loess did not conform to the single fractal distribution characteristics, whereas the overall particle distribution had fractal characteristics.The The non-uniform degree of mineral particles in the two soils was as follows: carbonate minerals in lime-treated loess > carbonate minerals in undisturbed loess > quartz minerals in lime-treated loess > feldspar minerals in lime-treated loess > feldspar minerals in undisturbed loess > quartz minerals in undisturbed loess.It can be seen that the non-uniformity of mineral particles in lime-treated loess was relatively more obvious than that in undisturbed loess, which is consistent with the observation of multi-scale microscopic images and the results of mineral particle size distribution curves. The analysis in this work can better provide a basis for the future study of the different soil mechanical properties of undisturbed loess and lime-treated loess, and also provide a method for the study of the mechanical properties of different types of soil.