The requirement of continuous and stringent growth on filtration performance, including longer life span, higher overall efficiency, lower initial pressure drop and more cost effective, has still drove filter media manufactures to research and develop. One of the possible way to achieve these challenges, was to utilize a dual-channel head-box with two sets of pulp conveying system, which can produce filter media with bulky and gradient properties. In this study, three kinds of commercial cellulose were chosen to make two layers filtration media, analyzed the effect of fiber blend on physical properties and filtration performance. By fine-tune the slurry ratio of top layer, we made one single layer and two layers composition filter media, the thickness and air permeability of composition media were higher than single layer media. According to ISO 5011, filtration performance test has been done to compare single layer media with composition media, this composition gradient profiles that provided the life span 37.0% improvement to the terminal pressure drop during dust injecting, and the dust hold capacity improved 34.7%, the main contributor of dust hold capacity was decided by top layer, however, the overall efficiency was depended on wire side layer. The air filter element was of essential, applied to purify the input air and prevent airborne dust from being sucked into to wear out and shortened the life span of the engine2. From the filter media manufacture point of view, the continuous challenge was to develop higher filtration performance filter media, but competitive price3. It has already well established in previous literature that multi-layer technology had unique and advantages to result in gradual and relatively slow increase in pressure drop and enlarge engine life time, especially can improve dust hold capacity obviously. Tucny4 studied via numerical simulations that clean without fouling process multilayer filter showed limited impact on filtration performance, however, Kaukopaasi5 has already confirmed in pilot scale trial that multi-layer media performed better than one single layer media, by which could reduce cost at same filtration performance level using cellulose instead of specialty fibers. Zhang et al.6 facilely fabricated multilayered needle-punched/melt blown composite nonwoven filter with good filtration performance regarding on high efficiency, low pressure drop and long service life, which can be good candidate for the removal of PM 2.5. Ling et al.7 prepared multilayered nanofiltration membrane by silk nanofibril and hydroxyapatite for water purification with high flux.With the rapid growth of automobile ownership globally every year, huge amount of filter elements, has already consumed plentiful forest and fossil resources to produce9. Nanofiber layers working as dust intercept layer fabricated onto supporting layer can enhance filtration performance, the loading dust gathered on nanofiber layer rather than penetrated into filter media, which was benefit for self-clean filter element11. Long et al.12 prepared submicro-fiber composition medium by dual-layers machine, found that after field test, pressure drop of wet-laid submicro-filter was about 45% lower than commercial filter medium and its filtration mechanism was surface fiber capture particles rather than pores. Depth filter media usually composed more than two layers and the potential biggest advance was dust hold capacity improvement6. By fabricating of two and three layers of filter media by melt blown and thermal bond fibers, Hasolli13 evaluated that top layer made of large fiber size and open fibrous structure, contributed to collect large size particles, and final layer was made of small diameter fibers and low porosity, contributed to remove fine particles. Electro-spinning and micro glass fiber also can be used to prepare composite filter media to enhance filtration efficiency15. However, these non-renewable and high cost fossil materials were used to fit these advanced methods can be damaged during pleating process, so these methods made air filter element more complicated and expensive for automobile filtration market16.Two kinds of multilayer filtration theory were researched for air intake filtration: depth filtration and surface filtration17. The obtained filtration media was tested according to ISO 501118, it was found that the life span to desired pressure drop improved 37%, and dust hold capacity increased 34.7%, the overall efficiency of all the samples were more than 99.5%, which can meet original equipment manufacture’s requirement.To the best of my knowledge, there was no article using all commercial natural wood cellulose to design two layers filtration media. To well meet the demand of the filter element market, 100% commercial wood cellulose was used to design and make composition media by lab-scale standard dynamic paper sheet former machine to design two layers air filtration media, which was the most suitable for current practical production. The paper mill only needed to prepare one more pulp conveying system and one plastic plate in head box to separate the slurry of top layer and wire side layer to produce dual-layer composition filtration media, like Fig. 2, and wire side layer’s base weight was 90 g/m2. The recipe of top layer was adjusted to make open fibrous structure with wide diameter and long length fiber, which was supposed to intercept large size dust13. The fiber blend and the resulted data were listed in Table Three samples, include two composition double layers (Nos. 4 and 5) and one single layer (No. 6) filter media were made with specific fiber blend for each layer to analysis how to effect filtration performance. For the composition media, we designed top layer’s base weight was 30 g/mNos. 3 and 6 were one single layer filter media sample, the recipe ratio was pulp 1:pulp 2:pulp 3 = 1:1:1, we can conclude the base weight was positive linearly related with paper thickness, and air permeability was inversely proportional with base weight. During the spraying process, the slurry was deposited onto forming wire round and round smoothly, so the thickness was going higher accordingly.Nos. 1 and 2 filter media were controlled as same base weight, however, pulp 1 and pulp 2 were used quantity equally for No. 2 media, and pulp 1 and pulp 3 quantity equally for No. 1 media, therefore, their thickness and air permeability were different because of the air permeability of pulp 1 was higher than pulp 2. From Table 3/h normally, and the normal filter element’s extended area was roughly 16 m2, so we set up face velocity value 5.33 cm/s for heavy duty truck, also same logic with light duty car to get close with practical application environment. From Fig. 19.The initial pressure drop of filter media was tested at face velocity 11.1 and 5.33 cm/s, respectively. Because the currently rated flow of commercial heavy duty truck was 720–1920 m3, 100cm2 sample was placed between two clamps, we can read initial pressure data online at the first 30 s before dust launched.The pressure drop was tested by Palas 3000, according to ISO 5011, the mass concentration of standard SAE fine A2 dust was adjusted as 1000 mg/m2 but different recipe, No. 4 was more bulky, their wire side were same as No. 3 regarding base weight and recipe, so that the pressure drop were mainly determined by compact layer. Comparison of one layer sample No. 6 and composition samples No. 4 and 5, we can concluded that composition media was benefit to decrease initial pressure drop, which was useful for fuel saving and power strengthening20.Pressure drop was decreased with increasing air permeability, the air permeability of Nos. 1 and 2 samples were much higher, we can see the pressure drop data was almost same. The samples of Nos. 3 and 6 were same recipe with different base weight, their pressure drop was inversely proportional with air permeability. No. 6 sample was formed as one layer, which means its top layer and wire side layer’s fiber blend were same, however, Nos. 4 and 5 sample were composition media, the base weight of these two sample were all 30 g/m3 to the untested sample, the initial pressure drop can be read at first 30 s, the termination of the differential pressure was set to add 2000 pa to the initial pressure. The initial pressure was mainly effected by air permeability as talked above, during the process of testing, dust was continuous sprayed onto the filter media, the dust load amount was liner increased, however, we can found from Fig. When Palas machine started running to test, the dust generator ejected specific gravimetric dust mass concentration 1000 mg/m21.The overall efficiency of filter media was determined by wire layer, which was higher density made of small diameter fiber, from Table 22.The fractional efficiency of particle size from 0.305 to 12 μm was conducted by two Welas 2070 in-line particle counter, located at upstream and downstream of filter media, respectively, to calculate the number of various size of particle before and after passed through the filter media. As shown in Fig. The filtration performance of composition media made by lab scale paper machine showed 4–6% higher thickness and 10–17% higher air permeability at same base weight compared with single layer media, which mostly attributed to the top layer modified by specific fiber blend. The unique characteristic of bulky structure caused 37.0% enhancement of longer life span and excellent filtration performance regarding on dust hold capacity improved 34.7% and overall efficiency was all kept more than 99.5%. The filtration efficiency was determined by mean pore size, and the fine and course dust can pass through filter media mainly happened in the early stage of testing, at the first 69 s, the fine dust can pass through the media, after 321 s, more than 90% of fine dust can be intercepted by interception between the fiber itself and formed layer.In order to obtain various properties of air filtration paper, we chose 3 kinds of raw pulps. Their basic physical properties as followed in Table The thickness of hand sheet was measured according to TAPPI T411, the air permeability was tested according to ASTM D737 under the pressure 200 Pa, the maximum and mean pore size was tested by capillary pore diameter measuring instrument  according to ASTM F316, the surface tension of infiltrating fluid was 16 mN/m. The filtration performance was tested by PALAS MFP 3000, according to ISO 5011 at the face velocity was 5.33 and 11.1 cm/s, respectively. The terminal pressure drop was 2000 pa. The dust hold capacity was gravimetric weighted before and after dust loaded, the filtration efficiency was conducted by two particle counter, and initial pressure can be read online at first 30 s from dust depositing. The morphology of resulted hand sheet was observed by scanning electron microscope .Standard dynamic paper sheet former machine  was used to simulate the two sets of slurry conveying systems in head-box of the paper machine. The specific fiber blend of wire side layer was prepared and sprayed onto forming wire, and then top side layer slurry was sprayed and deposited on top layer. The each layer and total base weight was designed and controlled by slurry amount, after wet paper web formed, the filter paper was vacuum dried and stored at constant temperature and humidity room for test.


Though recycling of waste paper is widely practiced but usually it is downgraded to lower valued recycled waste paper. Based on this concern, we report the development of novel NaOH/urethane aqueous system for extraction of microfibrillated cellulose from waste paper. The purity of so obtained microfibrillated cellulose (MFC) was evaluated by morphological tests using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and by evaluation of physicochemical properties using Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Morphologies of MFC studied by SEM and TEM showed that the size of purified cellulose fibrils reduced when compared to that of waste paper but fibrils are cleaner and smoother due to the removal of talc and lignin. XRD analysis revealed that MFC exhibits good crystallinity. The utility of sulfonated and pristine microfibrillar cellulose in removal of lead from contaminated water is also reported. Our results show that renewable, sustainable, cheap, and waste biomass like waste paper can be used for producing valuable second-generation high-value products. Amongst these, synthetic and natural polymers play critical and ubiquitous roles in our everyday life. However, due to the growing concerns about end-of-life disposal of petroleum oil-based synthetic polymers, alternate biodegradable and biosourced plastics are rapidly gaining considerable interest. Amongst these, cellulose, the most abundant naturally occurring amphiphilic polymer in the biosphere, with an estimated annual production of 7.5 × 10× 1012 t , sensors× 1012 t , etc. HoDespite the fact that cellulose is widely available, its insolubility in industrial solvents due to the extensive intrinsic hydrogen bonding, which forms a strong three-dimensional semicrystalline β-(1→4)-linked glucosyl units linear homopolysaccharide networks, makes its extraction difficult . Traditi2 groups is very effective in dissolution of cellulose in alkaline aqueous systems, we postulate that urethane, an ester of carbamic acid with a lone –NH2 moiety but considerably more reactive, can also efficiently dissolve cellulose in NaOH aqueous systems.The purpose of this investigation is to report our discovery of NaOH/urethane aqueous system to extract microfibrillated cellulose from waste paper. It is widely reported that urea with two –NH−1. The spectra were acquired with a resolution of 4 cm−1 and a total of 32 scans. 1H-NMR spectra were recorded on a Bruker DRX-400 spectrometer  at 400 MHz in CDCl3 solution. The chemical shifts were reported in parts per million (ppm) and with tetramethylsilane (TMS) as an internal standard. X-ray diffraction (XRD) patterns were recorded on Rigaku D-max diffractometer .Waste paper was collected from our office waste basket, whereas reagent-grade urethane , lead nitrate , p-toluenesulfonyl chloride , dimethyl acetamide , lithium chloride , and sodium hydroxide  were purchased from Sigma-Aldrich, Seoul, Korea, and were used as received. Waste paper was first shredded and boiled in deionized water for 30 min at 70 °C, washed with water to remove impurities, and then, dried overnight in a vacuum oven. NaOH and urethane were separately dissolved in deionized water to obtain 10 wt% urethane and 10 wt% NaOH aqueous solutions and subsequently mixed in deionized water to obtain 10 wt% NaOH/10 wt% urethane aqueous solutions. The dried waste paper pulp was added to the above NaOH/urethane solution, and the mixture was stirred for 180 min at room temperature after which, the reaction was quenched by adding cold deionized water to the reaction mixture and filtered by a vacuum filter. The solution was centrifuged at 5000 RPM for 30 min and subsequently dialyzed with deionized water until a constant pH was reached. The morphology was tested with a field-emitting scanning electron microscope (SEM)  and transmission electron microscopic (TEM) images were recorded on TALOS F200X , at operating voltage was 10 and 200 kV, respectively, in SEM and TEM. Fourier transform infrared spectra, FTIR, were recorded by a Thermo Scientific Nicolet iS10 Spectrometer  with and attenuated total reflection (ATR) accessory, in the wave band range from 4000 to 400 cm3)2 in the background electrolyte at a controlled pH of 5.5 maintained by adding 0.01 M NaNO3. The choice of slightly acidic condition was chosen based on the fact that real-life experimental soils are in this pH range. In a typical experiment, a microfibrillated cellulose films (1 g L−1) were added to the solution and then, vigorously stirred at 400 rpm using a magnetic stirrer. The amount of lead adsorbed was measured by EDTA titration method as reported by Hisada et al. [Batch type equilibrium adsorption experiments were performed using 0.1 mM Pb(II) solution prepared by dissolving Pb was estimated according to TAPPI 249-75 procedures. Briefly, 5 g of extracted microfibrillar cellulose was mixed with 500 mL of 7.5 wt% NaOH solution and stirred at 25 °C for 60 min. Precisely, 10 mL of 0.5 N potassium dichromate aqueous solution and 50 mL of concentrated sulfuric acid was added to 25 mL of the filtrate and stirred for 15 min. Finally, 50 mL of water and 2 drops of ferroin indicator were added and titrated against 0.1 N ferrous ammonium sulfate solutions until a purple color was obtained. For determination of holocellulose, 2 g of the sample was placed for 120 min in an oven maintained at 150 °C The percentage of α-celluloses, holocellulose, and lignin content were determined by the following equations:These platy and layered structure impurities are probably talc and other clay materials that are added in traditional paper making to reduce tackiness and increase hydrophobicity. After treatment with our NaOH/urethane aqueous system, SEM micrographs indicated that these inorganic impurities were effectively removed, which resulted in effective defibrillation of the cellulose fibers, leading to formation of micrometer long, ribbon-shaped cellulose fibers. + and Na+ form ionic hydrates whose size and concentration depends on the solution temperature. However, in order to extract cellulose, mere swelling is not sufficient and the cellulose must dissolve. This swelling is resultant of partial dissociation of the C6-OH moieties of cellulose molecules in NaOH aqueous solution. The binding of Na+ ions to hydroxyl groups of cellulose ruptures the intermolecular hydrogen bonds in cellulose and leading to the dissolution of cellulose. This phenomenon is augmented by adding carbamates like urea, thiourea, and urethane, as in our case, wherein the highly polar NH2 groups of urethane act as hydrogen bonding donor and receptor between NaOH and C6-OH to form new hydrogen bonds, which leads to decrease in crystallinity thereby changing the molecular confirmation of cellulose from semistiff rigid structure to extended wormlike flexible chains. The chemical composition of extracted fibers as determined by the procedure described in The mechanism of formation extraction of cellulose by our NaOH/urethane system can be explained on the basis of formation of inclusion complex of cellulose in alkaline carbamate aqueous solutions . It is w2O; 4H2O) [In order to understand the dissolution of extraction of microfibrillated cellulose by our NaOH/urethane aqueous system, DSC study was carried out. O; 4H2O) . In presO; 4H2O) , respect2 groups of cellulose backbone was slightly shifted from 2920 to 2900 cm−1 in NaOH/urethane-treated waste paper, which can be attributed to the partial removal of lignin and increase in the cellulose component as evident from the sharpening of asymmetric and symmetric methyl and methylene stretching groups present in the spectra of typical cellulose fiber-rich components. Besides this there was a drastic decrease in the intensity of the 1650 cm−1 peak, which can be assigned to stretching vibrations of aromatic C=C of lignin component in NaOH/urethane treated waste paper that further confirms our claim of removal of lignin by our alkali/urethane treatment. In typical alkali/carbamate system, though it is widely believed that NaOH interacts with hydroxyl groups of cellulose, the exact role of carbamates in cellulose dissolution is still not clear. It is believed that carbamate might play dual role of direct interaction with cellulose and/or influences the interaction between cellulose and the solvent. The chemical composition of paper is cellulose, hemicellulose, and lignin, and there are reports which suggest that the main characteristic peak of hemicellulose can be observed around 1728–1731 cm−1 [−1, which was assigned to acyl-oxygen CO-OR stretching vibration in hemicelluloses prominent in the case of waste paper, whereas in NaOH/urethane-extracted microfibrillar cellulose, the intensity of this peak was reduced marginally, which indicated that a portion of hemicellulose could also be removed by our newly developed system.The FT-IR spectra of untreated and NaOH/urethane-treated waste paper is plotted in 731 cm−1 . HoweverD) was calculated to be 79.36% and cellulose crystallite size as calculated by Scherrer formula was 6.9 nm, which further confirmed that our newly developed NaOH/urethane aqueous system was capable of producing highly crystalline microfibrillar cellulose. The thermal stability of microfibrillar cellulose extracted by our NaOH/urethane aqueous system as measured by TGA is illustrated in The X-ray diffraction spectra of waste paper and microfibrillar cellulose extracted using our NaOH/urethane aqueous system is plotted in 3H moieties on cellulose surface. The FTIR spectra of pristine and sulfonated microfibrillar cellulose plotted in −1 confirming the formation of –SO3H moieties on cellulose surface. The degree of grafting (DOG) was measured by weight gain method [a−Wo)/Wo) × 100 where Wa and Wo are the weights of membrane after and before treatment, respectively. From the weight gain analysis, the DOG was measured to be 8.9 wt%. Batch adsorption experiments performed to investigate the adsorption of Pb(II) on sulfonated microfibrillar cellulose films at increasing concentrations from 0.001 to 0.06 mg/L is plotted in 3H moieties, which provide active sites for capture of positively charged Pb(II) ions and retain the adsorbed lead moieties on these anchoring points. The magnitude of adsorption was higher at lower concentrations with both pristine MFC and waste paper showing almost the same adsorption efficiency, however, with increasing concentration of lead, sulfonated MFC showed better adsorption capacity. This result successfully proves that sulfonated microfibrillar cellulose can be very effective for removal of positively charged heavy metal ions from contaminated waters.Lead ion (Pb(II)) is the most common in drinking water due to widespread use of lead-based pipes used in domestic piping. Consumption of lead above the permissible level of 0.015 mg/L causes serious health problems like kidney malfunction, blood loss, cancer, etc. Although many techniques including membrane filtration, ion exchange, precipitation, adsorption, oxidation, reduction, etc. are in use to remove Pb(II), the most effective and economic method is adsorption. Many adsorbents both organic and inorganic can be used for removal of lead by adsorption, but naturally occurring polysaccharides and polysaccharide-derivatives like pectin , lignin n method  by weighIn summary, we report the discovery of NaOH/urethane aqueous solvent system for extraction of microfibrillated cellulose from waste paper. Morphological studies by SEM and TEM showed that tape structured, micrometer long cellulose fibers can be obtained using our newly developed technique. The excellent quality of microfibrillated cellulose fibers was confirmed using NMR and FTIR studies, whereas structural analysis by XRD showed that the cellulose fibers obtained by our technique were highly crystalline with 79.36% degree of crystallization and average crystallite size of 6.9 nm as calculated by Scherrer’s equation. The utility of pristine and sulfonated microfibrillated cellulose for removal of lead from contaminated water was also measured that showed almost 250% increase in adsorption capacity of lead by simple sulfonation.


Eruca sativa  seeds for six months on board the International Space Station resulted in delayed seedling establishment. Here we investigated the physiological and molecular mechanisms underpinning the spaceflight effects on dry seeds. We found that ‘Space’ seed germination vigor was reduced, and ageing sensitivity increased, but the spaceflight did not compromise seed viability and the development of normal seedlings. Comparative analysis of the transcriptomes (using RNAseq) in dry seeds and upon controlled artificial ageing treatment (CAAT) revealed differentially expressed genes (DEGs) associated with spaceflight and ageing. DEG categories enriched by spaceflight and CAAT included transcription and translation with reduced transcript abundances for 40S and 60S ribosomal subunit genes. Among the ‘spaceflight-up’ DEGs were heat shock proteins (HSPs), DNAJ-related chaperones, a heat shock factor (HSFA7a-like), and components of several DNA repair pathways . The ‘response to radiation’ category was especially enriched in ‘spaceflight-up’ DEGs including HSPs, catalases, and the transcription factor HY5. The major finding from the physiological and transcriptome analysis is that spaceflight causes vigor loss and partial ageing during air-dry seed storage, for which space environmental factors and consequences for seed storage during spaceflights are discussed.In the ‘Rocket Science’ project, storage of  Plant science research during spaceflight, including on orbiting space platforms such as the International Space Station (ISS), has revealed novel mechanisms and the potential of ‘dry’ seeds, growing seedlings, and flowering plants to respond to environmental change and extreme conditions outside their evolutionary history ,2,3,4,5.In many organisms, including most plants, the passage through arrested life stages is associated with desiccation tolerance, the ability to survive extreme water losses by expressing protection mechanisms for the low-hydrated tissues and macromolecules in the ‘air-dry’ state ,20,21,22Arabidopsis thaliana (Arabidopsis) and tobacco seed lots exposed for >>1 year to the outside space of the ISS  showed delayed germination (reduced germination speed), but with ~90% viability when shielded from short-wavelength ultraviolet (UV) radiation (>1 GJ m−2 UV110–400 nm) [5 Pa), 1−3 µGy day−1) confirmed that ~1 GJ m−2 UV254 nm is indeed a lethal dose for Arabidopsis and tobacco seeds but not for field bindweed seeds, which have a hard seed coat with phenolic pigments potentially providing the protective shielding against the UV radiation. The finding that shielding against short-wavelength UV radiation retains viability was also found to be true for dry seeds of other plant species and for desiccation-tolerant bacterial and fungal spores; also, there seem to be species-specific differences in the self-shielding properties by phenolic pigments in seed and spore coats  were sent to LEO aboard Soyuz TMA-18M to spend six months on board the ISS, returning to Earth on 2 March 2016, as part of the ‘Rocket Science’ project [On 2 September 2015, 2 kg of rocket seeds  were used in the Rocket Science project [Seeds of salad rocket,  project . CompanyFor the space journey , Space sSeeds were transferred to 2 mL Eppendorf tubes and exposed to 70% RH  for three days at 22 °C and then exposed to 35 °C for the specified number of days (length of CAAT). Seeds were then dried above silica beads at 35 °C for 2 h and subsequently stored in sealed vials at room temperature until germination assays were carried out. CAAT is a commonly used method in seed science and industry to compare the ageing sensitivity of seeds during dry storage. To achieve this in an experimentally reasonable time, the combination of increased temperature  and RH  is required ,22,37.−1 m−2) in a Panasonic MLR-352 Environmental Test Chamber  at the temperatures indicated. In order to conduct the thermal time modeling  – conductivity of control solution (µS cm−1)) / weight of replicate (g).Four replicates of 100 seeds of both Space and Earth seeds were weighed and added to 100 mL beakers containing 50 mL distilled water and stirred to ensure that all seeds were submerged. Beakers were covered and placed at 20 °C. Electrical conductivity (EC)  of solutTotal RNA was extracted from dry or CAAT seeds for each of Earth-WS, Earth, and Space seed batches, three replicates per treatment, as outlined previously . Its quaEruca sativa (NCBI KF442616.1), chloroplast  and ribosomal  sequences of closely related organisms. To remove read contamination, DIAMOND [E. sativa) in forward reads were removed by filtering forward reads with 32% to 39% GC content and their respective pairs. Trimmed and filtered read pairs were used to run a de novo transcriptome assembly using Trinity [https://www.blast2go.com) [Sequencing quality was checked with FastQC version 0.11.05 . Read qu DIAMOND  version a NCBI KF2616.1, c Trinity . Assembl Trinity . Gene on2go.com)  with ca.10(p)) using one minus Pearson’s correlation coefficient and visualized in Morpheus [Read pairs, used for de novo transcriptome assembly, were mapped against the longest assembled transcripts using GSNAP with the additional parameters -N 1 and --npaths 7. Subread feature Counts version 1.6.2.  was usedMorpheus . g) for 3 min in a microcentrifuge to facilitate the formation of phase separation. The lower organic layer (hypophase) containing the isoprenoids was recovered and transferred to a fresh 1.5 mL tube, and the aqueous phase was re-extracted with an equal volume of chloroform. The combined organic phases were dried under a stream of N2 gas. UPLC-PDA was used to separate, identify, and quantify the pigments. The dried samples were re-dissolved in ethyl acetate (50 µL) and centrifuged at maximum speed for 5 min to remove potential precipitate from the sample. The clean extract (30 µL) was transferred to a ULPC glass insert previously mounted in a glass vial and capped, from which 3 µL was injected onto the UPLC. Chromatographic separation was carried out in a reverse phase mode using a C18 column  and a linear gradient of mobile phases A ) and B (acetonitrile (ACN)/ethyl acetate ). The column operated at 30 °C with a flowrate of 0.6 mL/min; automated sample temperature was kept at 8 °C. Detection was performed using a UV/Vis scan ranging from 200 to 600 nm. Comparative absorption spectrum and retention times with authentic standards were used to identify individual carotenoids, chlorophylls, and tocopherols [The carotenoid and chlorophyll pigments and antioxidant tocopherols were extracted and quantified as described in Drapal et al. . Expandiopherols . QuantifEruca sativa (rocket) from the Brassicaceae plant family were used in the experiments. The ‘Earth-WS (Warehouse Stock)’ ground control seed batch that had neither been packaged nor flown in space was used to establish the temperature dependence of rocket seed germination (50%), which is the inverse of the average time to 50% germination (t50%) of the seed population. Thermal time modeling revealed a maximum germination percentage of 95–100% over a wide range of temperatures (7 to 35 °C), indicating that seeds were nondormant. For the Rocket Science project [Seeds of mination . Followimination B. Testa mination B and submination D. Germin project , rocket The three seed batches, Space (blue packets), Earth (red packets) , and Eartop panel), on average reaching 50% germination 1.1 h later. When artificial ageing was applied, 15 days of CAAT increased the time to reach 50% germination (t50%) of Earth-WS and Earth seed batches by 34.4 and 26.6 h, respectively (bottom panel). However, the t50% of the Space seed batch was most affected by the 15 day CAAT, being increased by 46.4 h (bottom panel). The Space seed batch was already significantly more susceptible to CAAT at eight days of CAAT  on germination was therefore analyzed on all three seed batches: Earth-WS, Earth, and Space seeds . The CAA of CAAT B. The doectively B. Thus, Seed ageing during storage reduces seed quality initially by reducing the vigor (speed and uniformity of germination) and only subsequently by reducing the viability of a seed population . ConsistIn light of the effects of spaceflight on seed germination kinetics, the transcriptomes of Space and ground control (Earth and Earth-WS) seed batches were investigated to see if transcript abundance changes may suggest mechanisms by which spaceflight caused the reduced germination vigor and increased the ageing sensitivity. Further, because the difference between germination of the ground control and Space seed batches was largest following 15 days CAAT, their transcriptomes upon ageing were comparatively investigated. This revealed potential ageing-related processes involved in the effect of spaceflight and allowed determination of whether the ageing response of space-flown seeds differs to ground control seeds. Thus, the transcriptomes of the three seed batches , ‘dry seed’ without or with 15 days CAAT, were comparatively analyzed . E. sativa genome was not available, a de novo assembly was used as a reference for transcript quantification. Following filtering steps, the expression results for 26,234 transcripts were obtained and are accessible via the Rocket Science Gene Expression Viewer  between pairwise comparisons of treatments were identified using NOISeqBIO . In geneNotably, Space seeds exhibited fewer DEGs than the ground control  seeds following CAAT treatment , and the‘Down’ indicated by arrows). However, some terms were more strongly associated with a reduction in gene expression following either spaceflight or ageing. For example, terms in cluster II such as ‘seed oilbody biogenesis’ were more associated with a reduction in gene expression following CAAT. On the other hand, the term gene expression and others in cluster III were more associated with a reduction in expression following spaceflight (‘Up’ indicated by arrows). For the GO term ‘response to osmotic stress’ in cluster V the pattern was a bit more complicated, as this GO term was enriched in both genes that were associated with an increase in expression and genes that were associated with a decrease in expression following CAAT. This suggests that a number of genes belonging to this GO term category were induced by CAAT and a number of other genes belonging to this GO term category were repressed by CAAT. Cluster VI represented GO terms that were mostly associated with genes that increased in expression only following spaceflight and included terms such as ‘response to stress’, ‘response to light’, and ‘response to radiation’  term enrichment analysis was performed . Similarceflight . Conversdiation’ . AlternaTo investigate the processes highlighted by the transcriptome analysis in more detail, we assessed a number of DEGs with described functions belonging to the identified GO terms . One of RAP2.3, which follows a similar trend in decreasing due to CAAT and spaceflight  and chaperones ,60,61 wenscripts  and othenscripts . For exath seeds . Again, ce seeds  and HSFAs  also functions in DSB repair, as well as in other pathways, including the base excision repair (BER) pathway [LIG1 was significantly induced by CAAT by ca. 35% in all seed batches and was also 14% higher in Space as compared to Earth dry seed  detoxification  [Up in Space patterns in the GO terms ‘response to radiation’ and ‘response to light’  ,72 were ductase)  were indo light’  were at n in HY5 .In the Rocket Science project, a spaceflight including six months on board the ISS in low Earth orbit (LEO) had a subtle, but significant, effect in delaying germination of rocket seeds, consistent with the observations of over 5000 schools across the UK, which also reported lower seedling survival in space-flown seeds at 17 days and reduced seedling height at 21 days . The majE. sativa, are commercially important salad crops cultivated worldwide [Rocket species, including orldwide . It is torldwide ,15,16,17orldwide ,4,5. TheEruca species are adapted to the Mediterranean region [The space factors of gas, pressure, and humidity are also unlikely to play a major role in our Rocket Science experiment because the seeds were stored in a low-hydrated state in hermetically sealed packs (at 25% RH) inside the ISS A. Duringn region , have thn region . Mechanin region  or impron region  seed gerShielding provided by the ISS during the six months of rocket seed storage in LEO excluded the exposure to short-wavelength UV radiation known for its negative effects on seed lot viability ,30,31. ETo determine if ageing and space travel shared any parallels in terms of their effects of germination physiology and the transcriptome, RNAseq analysis of dry and controlled artificial ageing treatment (CAAT) rocket seed samples were performed. Both the physiological assays and transcriptome analysis pointed towards a potential partial ageing effect of the spaceflight. Compared to the CAAT, the effects of spaceflight were relatively subtle. For example, the delay in germination caused by spaceflight was less than the effect of eight days of CAAT but greater than the effect of four days of CAAT. Most intriguing for the global changes was that the space-flown dry seed transcriptomes were positioned between the dry seed ground controls and the CAAT samples in the PCA analysis A. OverlaEruca sativa seeds contain >40% oil, and the 8.0% overall moisture content of the Space seeds could therefore provide such pockets of higher moisture content distinct from Earth seeds (6.3%) during the various stages of the spaceflight and seed packaging process. Selective degradation of transcripts has also been shown to contribute to transcriptome differences in dry seeds owing to the distinct relative stability of transcripts [Transcriptome analyses of dry-stored seeds have been conducted over various storage periods and ambient conditions  to investigate their relation to after-ripening and dormancy release as well as longevity, ageing, and viability loss. Long-term seed storage experiments  revealed that in many cases RNA integrity  of dry seeds decreased over storage time before viability loss . Comparenscripts ,88,89. Inscripts ,21,71,73nscripts . Irradianscripts . We founSimilar to our CAAT, which achieved rocket seed ageing within 8–15 days , others used elevated RH and temperature to study the transcriptome and proteome changes during their short-duration artificial seed ageing treatments ,92,93. EATM transcript abundance is higher in Space samples and increases in Earth samples upon CAAT (XPB (DNA ligase 1 (LIG1), involved in DSB and single-strand break repair [LIG IV and LIG VI, which are associated with DSB repair in seed longevity and elevation in expression following seed imbibition, were not affected at the transcript level. Some components of the photoactivation repair (PR) pathway are also upregulated in rocket seeds upon CAAT, but except for HY5 no elevated transcript abundances were evident in dry Space seeds (ATM) and NER (XBP) DNA pathway transcripts . Reppon CAAT . For theAAT  during long-term warehouse storage is a major challenge for the global seed industry and is achieved by reducing humidity, temperature, and oxygen to slow down the rate of seed ageing in the dry state. The resilience of dry seeds was revealed in exposure experiments to the harsh space environment in which their viability was maintained for a considerable time if shielded against the short-wave UV irradiation. Our Rocket Science project results suggest that for maintaining the vigor component of the crop seed quality during long-term spaceflights, additional protection is required. We hypothesize that the observed rocket seed ageing during the space travel, which included six months of dry storage on board the ISS, is caused mainly by exposure to low-level ionizing radiation from galactic cosmic rays, trapped protons, and solar energetic particles, perhaps in combination with mechanical vibration. The absorbed radiation dose on board the ISS would equate to ~50 mGy, >>100-times more than on the Earth surface ,75,80, a


Verticillium dahliae and Macrophomina phaseolina). Overall, the plants had a distinctive and genotype-dependent rhizosphere microbiome with higher abundances of known beneficial bacteria such as Pseudomonads and Rhizobium. The rhizosphere microbiome played a significant role in the resistance to the two soil-borne pathogens as shown by the differences in microbiome between high and low resistance cultivars. Resistant cultivars were characterized by higher abundances of known biocontrol microorganisms including actinobacteria  and unclassified acidobacteria , in both pathogen trials. Additionally, cultivars that were resistant to V. dahliae had higher rhizosphere abundances of Burkholderia and cultivars resistant to M. phaseolina had higher abundances of Pseudomonas. The mechanisms involved in these beneficial plant-microbial interactions and their plasticity in different environments should be studied further for the design of low-input disease management strategies.Microbial-root associations are important to help plants cope with abiotic and biotic stressors. Managing these interactions offers an opportunity for improving the efficiency and sustainability of agricultural production. By characterizing the bacterial and archaeal community (via 16S rRNA sequencing) associated with bulk and rhizosphere soil of sixteen strawberry cultivars in two controlled field studies, we explored the relationships between the soil microbiome and plant resistance to two soil-borne fungal pathogens ( However, understanding the complexity and drivers of the soil microbiome, remains a challenge, and the practical application of ecologically based agricultural management strategies not feasible4. Soil bacterial diversity and abundance is primarily regulated by small- and large-scale changes in abiotic factors such as soil pH, oxygen concentration, and the availability as well as quality of carbon (C)1. Large variations in these factors exist in the macro  and microenvironments  within a soil profile, producing hotspots of microbial diversity and activity. The rhizosphere, the volume of soil that is directly influenced by plant roots and which spans usually 1 mm from the root surface, can contain up to 30,000 bacterial and archaeal species5. Furthermore, the rhizosphere typically has higher microbial biomass and activity when compared to the surrounding bulk soil6. This microbial hotspot within soil habitats is driven by plant root presence, nutrient and water uptake as well as the continuous release of carbon compounds through root rhizodeposits8. Differential bioavailability and assimilation of carbon compounds by soil microorganisms results in the selective promotion of certain taxa and inhibition of others9.Soil microorganisms support key soil processes such as decomposition, mineralization, aggregate formation and biocontrol of plant diseases, all highly relevant to agricultural production10. Soil bacteria such as Pseudomonas, Bacillus and Rhizobium can increase the concentration of plant available phosphorus (P) in soil by releasing phosphatase enzymes or organic chelates11. Root-exudate production and the release of C compounds to usually C-deprived soil microorganisms, is known to trigger microbial activity and the production of exoenzymes which in turn further accelerates the decomposition of organic molecules in the rhizosphere and nutrient release13. As a result, rhizosphere soil typically has larger N mineralization rates and higher concentrations of bioavailable nitrogen (N) and P when compared to bulk soil, regardless of the plant species considered6. Additionally, certain strains of Bacillus, Pseudomonas, Burkholderia and Acinetobacter are thought to increase the availability and uptake of micronutrients such as Zn and Fe for mung bean, rice and corn16. Increases in plant growth have also been related to the induction of changes in plant root architecture and increase in root absorptive surface through the bacterially secreted plant hormones like indole-3-acetic acid, gibberellins and cytokinins17. Plants with adequate nutrition may be more resilient to biotic stress; in addition, rhizosphere microorganisms are known to have also direct impacts in plant health by triggering plant defenses10. Induction of systemic resistance in tomato plants by the bacterium Azospirillum brasilense was attributed to the capacity of this microbial strain to produce ethylene, a hormone involved in plant defense17. In tomato plants rhizosphere, bacteria from Bacillus, Pseudomonas, and Azotobacter triggered the production of antioxidant peroxidase and polyphenol oxidase enzymes, reducing the incidence of early blight disease by the pathogenic fungus Alternaria solani18. Finally, numerous bacteria have been identified that are antagonists of filamentous fungal pathogens such as Verticillium dahliae and are therefore able to directly suppress the pathogen via competition, production of antibiotic substances, siderophores, protein degrading enzymes and volatile compounds19.Root-associated microorganisms, either symbiotic or free living are key for plant nutrition and health and, because of this, the rhizosphere is frequently compared with the human gutFragaria x ananassa Duchesne ex Rozier) is a fruit crop highly appreciated throughout the world, and therefore of major economic importance20. World production of this crop increased sharply over the last decades reaching over 13 million tons in 201721. Currently, the largest producer is China, followed by the USA, Mexico, Egypt, Turkey and Spain21. Within the USA, California strawberry production accounted for 88% of the U.S. market in 2017, representing the 4th most valuable agricultural commodity in the state with a total revenue of 3.10 billion dollars22. Since the late 1950s, the California strawberry industry relied on the use of preplant soil fumigation with methyl bromide to control soil-borne diseases23. However, since the complete phase out of methyl bromide in 2016 under the Montreal Protocol due to its stratospheric ozone depleting nature, strawberry growers are facing the challenge of maintaining their productivity while using alternative disease management strategies24. At the same time, new diseases such as the soil-borne fungus Macrophomina phaseolina, are starting to emerge threatening the strawberry industry even further25. Other fumigants such as chloropicrin are currently in use, although their efficacy is substantially lower than methyl bromide26. Furthermore, urban development and encroachment in the areas with the most intensive strawberry production, severely limits the application of pesticides. The above-mentioned restrictions in fumigant use have shifted the interest of the industry towards integrated strategies combining cultural techniques such as anaerobic soil disinfestation, cover crops, or crop rotations to improve soil health, as well as the use of resistant cultivars23. To date, strawberry cultivars have been identified that are tolerant to soil-borne pathogens like Verticillium dahliae28 and Fusarium oxysporum f. sp. fragariae. Resistance to these soil-borne pathogens seems to be achieved through the expression of multiple genes29 thought to regulate the production of substances with antifungal properties such as catechin, caffeic acid, or citric acid30. Cultivars with different degrees of resistance to Macrophomina crown rot have also been identified, yet little is known on the mechanisms involved despite its recently increasing incidence in California and other regions of the world33. The interplay between roots and soil microorganisms is likely involved in the resistance to these soil-borne fungal pathogens, as it has been previously observed for cucumber, common bean and tomato36. Yet, to date no information is available on how the rhizosphere microbiome influences the resistance of strawberry cultivars to these pathogens. This knowledge is instrumental in the selection of strawberry cultivars that will allow the strawberry industry to maintain its high yields and competitiveness, while reducing the need for agrochemicals with the associated reduction in production costs and environmental impacts.Cultivated strawberry  characterize the rhizosphere microbiome in strawberry plants under commercial field conditions; (ii) identify changes in the rhizosphere microbiome in the presence of two soil-borne fungal pathogens (V. dahliae and M. phaseolina) and potential increases in the presence of beneficial rhizosphere microorganisms and (iii) evaluate the relationships between the rhizosphere microbiome and plant traits such as resistance against soil-borne pathogens, plant growth and nutrition. We hypothesized that strawberry plants have the capacity to recruit beneficial microorganisms and establish a rhizosphere microbiome different from bulk soil. Further, variations in plant traits derived from decades of breeding cultivars would also have an impact on belowground root traits and therefore different strawberry cultivars would show different microbial communities. Finally, plants with higher resistance will exhibit a different microbiome from plants with low resistance having a higher abundance of beneficial bacteria and archaea; resistance to soil-borne pathogens, growth and nutrient uptake, would be corelated with the composition of the rhizosphere microbiome.Here, we used two ongoing field trials established to determine the host resistance to A total of 3,939,353 high quality bacterial and archaeal sequences were generated across both the bulk soil and rhizosphere soil samples. After filtering chloroplast and unassigned reads, 3,786,463 sequences were clustered into 5,600 OTUs representing 33 Archaeal and 5567 Bacterial OTUs from 353 genera.Differences in OTU community structure between sample types (rhizosphere and bulk soils) in the two pathogen trials were examined via non-metric multidimensional scaling (NMDS) using a Bray–Curtis dissimilarity of the standardized and transformed data Fig. .Figure 1p = 0.001) .We observed significant differences in the soil microbiome between pathogen trials although the magnitude of the differences depended on the sample type  revealed that the rhizosphere and bulk soil samples differed by 17.87% and 13.56% in the oil Fig. a. Additioil Fig. a. In theoil Fig. b. The phoil Fig. b.Figure M. phaseolina trial than in the V. dahliae trial but remained similar in the rhizosphere soil across trials  was significantly higher in the bulk soil of the 19. The LEfSe analysis performed on samples from within in the V. dahliae trial data revealed 191 taxonomic clades that exhibited greater relative abundance in the bulk soil relative to the rhizosphere  to assess the differences in the relative abundance of the different microorganisms at the genus level, specifically focusing on a subset of well-known antagonists of filamentous fungal plant pathogensre Table , 6 of whre Table a. This are Table a. On samts Table . Additiots Table b.Figure M. phaseolina trial) or grown in soil with a high concentration of the fungal pathogen . To compare control vs inoculated plants, we aggregated all the plots, regardless of the cultivar. Thus, the control had a total of 10 replicates (plots), while inoculated plants had 40 replicates (plots) in each trial. The rhizosphere microbial community composition of plants exposed to V. dahliae was significantly different from control plants . An average dissimilarity of 27.11% in OTU relative abundances was found between infested and control plants based on SIMPER analysis (performed with taxa grouped at the genus level) in the V. dahliae trial. The genera found in higher relative abundance in the control plants include Arthrobacter, Steroidobacter, Pseudomonas, Azotobacter, Luteolibacter, Nakamutella, Saccharibacteria (genera incertae sedis), WPS-1 (genera incertae sedis), Micromonospora, Arenimonas. On the other hand, the archaeal genus Nitrosphaera and bacterial genera Gaiella, Hydrocarboniphaga, Rubrobacter, Streptomyces, Mucilaginibacter, Rhizobium, Pseudonocardia, Niastella and Aminobacter exhibited greater relative abundances in V. dahliae infested plants . SIMPER analysis (performed with taxa grouped at the genus level) revealed an average dissimilarity of 27.07% between infested and non-infested treatments. The bacterial genera Nakamurella, Gemmatimonas, Arthrobacter, Flavobacterium, Phycicoccus, Saccharibacteria (genera incertae sedis), Burkholderia, Bradyrhizobium, Novosphingobium, Luteolibacter, Arenimonas and Sphingobium exhibited higher relative abundances in the control relative to the inoculated plants and contributed at least 1% to the total dissimilarity between groups. The bacteria in the genera Pseudomonas, Isoptericola, Xanthomonas, Steroidobacter, Streptomyces, Mucilaginibacter, Gaiella and Massilia exhibited higher relative abundances in the rhizosphere of plants inoculated with M. phaseolina and contributed at least 1% to the total dissimilarity between groups , showing the cultivar-dependent selection of microorganisms. Similar results were observed for the 10 cultivars grown in the M. phaseolina trial . Furthermore, Shannon diversity in the rhizosphere microbiome was also found to be significantly different between cultivars in the V. dahliae trial , indicating the selection of microbes by cultivar in the rhizosphere depended strongly on the environmental conditions and the microbiome of the surrounding bulk soil.Four of the 16 cultivars were represented in both pathogen trials to test the stability of rhizosphere plant-microbial interactions and whether the selection of microorganisms would lead to similar rhizosphere microbial communities, regardless of the environmental conditions and bulk soil microbial community. Significant differences in the microbiome were observed among cultivars , three cultivars showing resistance between 50–70% (moderate) and two cultivars showing more than 70% mortality (susceptible)  Table . These des Table . Twelve rs Table , includiV. dahliae (p = 0.06), although the Shannon diversity index was significantly positively correlated to the contents of Ca (p = 0.04) and Mg in the leaves of the strawberry plants , a moderate , and a resistant group  . A SIMPER analysis on these cultivars (conducted with taxa grouped at the genus level) found an average dissimilarity of 28.8% between susceptible and resistant cultivars. A total of 20 genera contributed to at least 1% of the differences between resistant and susceptible cultivars  Table . No diffrs Table . Eleven rs Table , includiM. phaseolina trial (Table p < 0.001), and some other significant correlations were found between leaf K and leaf Ca, Mg and P, although none of these leaf nutrients were correlated to the rhizosphere microbial diversity (Shannon index). The dbRDA analysis revealed that plant biomass was significantly associated with the composition of the microbiome in the rhizosphere, but not with the plant leaf nutrient contents  in the case V. dahliae controls versus the inoculated plots, contributed to the observed differences in this field.We assessed whether the presence of the soil-borne fungal pathogens increased the abundance of beneficial bacteria in the rhizosphere of the strawberry plants. The presence of 41. However, comparison of the same cultivars grown parallelly in the two pathogen trials, showed that their microbial communities changed depending on the field where they were grown, suggesting strong genotype by environment interactions. In fact, these same cultivars showed very different leaf nutrient concentrations and mortality rates, that could be driven by the different soil-borne pathogens, initial soil fertility and microbiome in both pathogen trials60.Differences in rhizosphere microbiome between the cultivars showed genotype-dependent selection of the rhizosphere microorganisms as has been observed in other plant species61. Mendes et al.34 studied the rhizosphere microbiome of different common bean cultivars with different degrees of resistance to Fusarium oxysporum, and observed that a resistant cultivar had a higher abundance of certain bacterial families such as Pseudomonadaceae, Bacillaceae, Solibacteraceae and Cytophagaceae. Using a combination of 16S rRNA sequencing, shotgun metagenomics, and plate cultures, Kwak et al.36 were able to determine that the resistance of the tomato cultivar Hawaii 7996 to the soil-borne pathogen Ralstonia solanacearum was conferred by the presence of a Flavobacterium strain. In our study, comparisons in the rhizosphere microbiome between cultivars with different levels of resistance against the two fungal pathogens showed significant differences. Correlation analysis carried out through db-RDA showed no relationship between the whole community of the rhizosphere and plant mortality suggesting that resistance could be associated to the presence of biocontrol taxa but not general suppression of the soil-borne diseases. Taxa found in higher abundance in the cultivars resistant to V. dahliae included, Burkholderia, Sphingomonas and Novosphingonium which have been found to suppress this fungal pathogen in previous studies62. Additionally, higher abundances of unclassified Acidobacteria  and Actinobacteria  were found in resistant strawberry cultivars. Acidobacteria have been found to correlate well with apple tree growth before63 and to be negatively correlated to the abundance of Fusarium oxysporum in banana64. Arthrobacter is a known antagonist to V. dahliae through the production of cell wall degrading enzymes65, whereas Nocardioides and Gaiella have recently shown to be essential to the resistance of strawberry plants against F. oxysporum66. A recent study performed with soils collected at the continental scale, found that acidobacteria and actinobacteria are key players in the suppression of the fungal pathogen Fusarium oxysporum67. Results of this study show that both acidobacteria and actinobacteria are also key taxa in the resistance of strawberry cultivars against V. dahliae.Previous studies have shown that, cultivar-specific rhizosphere microbiomes are associated with increased resistance to soil-borne diseases. Resistance can be due to the presence of biocontrol taxa that directly or indirectly suppress the disease (specific disease suppression), and plant growth promoting bacteria that induce systemic disease resistance in the plant. However it is also possible that disease suppression is the result of the coordinated action of all the microorganisms present in the rhizosphere M. phaseolina trial, resistant cultivars were characterized by greater abundances of Pseudomonas, actinobacteria , which are well-known to be fungal antagonists and plant growth promoters68. Additionally, the rhizosphere of resistant plants was also characterized by higher abundances of unclassified Acidobacteria . Interestingly, the same taxa seemed to be in higher abundances in the resistant cultivars in the two field trials, showing a clear role of the rhizosphere microbiome and suggesting a common mechanism in the resistance to soil-borne diseases in the strawberry cultivars.In the V. dahliae field trial suggesting a role in the response to abiotic stress. The soil at the experimental fields had a high concentration of Mg and low Ca: Mg ratio that could create nutrient stress in the plants as shown by the significant correlations between leaf Mg, Ca, Ca:Mg with plant mortality. Together, these findings show a role of the rhizosphere microbiome in regulating the response of the plant to biotic and abiotic  stress. The specific mechanisms and plasticity of these beneficial plant-microbial interactions needs to be further investigated. In particular, given the strong genotype by environment interactions observed when cultivars were compared across the two fields, it is necessary to determine what are the optimal soil conditions and management strategies that can promote beneficial plant-microbial interactions.In addition to playing a role in plant resistance to biotic stress, the rhizosphere microbiome was significantly correlated to other plant traits including plant biomass and leaf Mg and Ca:Mg ratio in the Pseudomonas, and actinobacteria such as Arthrobacter, Nocardioides and Gaiella. Selection of rhizosphere microorganisms is strongly associated to the different genotypes and therefore seems to be a genetic trait that could be selected through breeding; nevertheless, the microbiome selected by a specific cultivar depends largely on the environmental conditions and microbiome of the surrounding soil. Thus, this suggests that soil health may be important in the establishment of these beneficial plant-microbial interactions.Strawberry plants can recruit microorganisms to their rhizosphere from the pool of microbes available in the soil, increasing the relative abundance of beneficial microorganisms involved in nutrient uptake and resistance to soil-borne diseases. Results of two field trials using 16 cultivars show a higher abundance of diazotrophic bacteria in the rhizosphere of strawberry plants which should be further explored as a strategy to reduce inputs of N fertilizers. Furthermore, cultivars resistant to the two soil-borne pathogens consistently showed higher abundances of biocontrol microorganisms such as Macrophomina phaseolina and Verticillium dahliae. These trials were established in October through July in 2015–2016, 2016–2017 and 2017–2018. Plant material for the current study was selected from the two pathogen trials established in October 2016. We selected 10 cultivars exhibiting a range of resistance, from high to low, to Macrophomina phaseolina and Verticillium dahliae. Out of the 10 cultivars, 6 were unique to each trial and 4 were grown in both trials, for a total of 16 different cultivars evaluate in this study. The names and breeding programs of the cultivars selected from each field trial are shown in Table Strawberry cultivars from different breeding programs and representing a range of resistance to soil-borne fungal diseases, were selected from two large ongoing field trials, evaluating 90 cultivars and elite selections for their resistance to V. dahliae trial had a pH of 7, electrical conductivity (EC) of 1.2, cation exchange capacity (CEC) of 25.5, and 3% organic matter. The soil in the M. phaseolina trial had a pH of 6.8, EC of 1.6, CEC of 17.4, and 3% organic matter.Two parallel (located 0.4 km apart) replicated pathogen trials were established on the California Polytechnic State University campus in San Luis Obispo, California (USA) to evaluate the 90 strawberry cultivars and elite selections for their resistance to the soil-borne fungal pathogens. The soil at both field sites was classified as Pachic Haploxeroll on a 0–2% slope with a clay loam texture. The soil in the The trials followed a complete randomized block design with four blocks. Briefly, in each trial, strawberry plants were grown in 1.6 × 2.5 m plots established in raised beds (0.3 m high). Plots contained 20 plants of the same cultivar and constituted the experimental unit. Ninety plots (one per cultivar) were arranged randomly within each block and 4 replicates (blocks) were established Fig. . This deV. dahliae trial relied on high levels of microsclerotia found naturally in the field soil (20 CFU g−1 soil). The Andersen sampler technique69 was used to estimate V. dahliae populations in field soil with the following methods. Eight soil cores (2.5 by 30 cm) were collected from each replicated plot, bulked, and thoroughly mixed for analysis. Soil aggregates from each sample were broken up and thoroughly mixed for 5 min. After air drying the soil at room temperature for 1 week, each sample was ground into a fine powder with a mortar and pestle. An Andersen air sampler was used to distribute the soil on the surface of NP-10 medium70. Three replicates of 5 total plates were processed for each sample. After 10 days of incubation in the dark at 20 °C, the surface of the agar medium was gently washed under a stream of water to remove soil particles, and microsclerotial colonies of V. dahliae were counted with the aid of a dissecting microscope. The estimate of soil inoculum density was expressed as an average number of CFU of V. dahliae g−1 of dry soil.Differences in pathogen history between the two field sites prompted distinct methods for ensuring host infection. The Macrophomina phaseolina was not naturally present in the soil at the field site and had to be inoculated. Prior to the inoculation, the field was fumigated (392 kg ha-1 of a 50% chloropicrin + 50% methyl bromide solution) to ensure the removal of all other soil-borne fungal pathogens. Fumigation was carried out in 2015, two years before the start of this trial, since the effects last usually 2–3 years. At transplanting, 5 g of M. phaseolina inoculum  was applied to the soil-crown interface. Macrophomina inoculum was produced from field isolates collected from diseased plants in 2014 and 2015 following procedure by Mihail71. Briefly, a homogenized 1:0.4:0.4 sand: cornmeal: deionized water mixture was autoclaved for one hour on two separate days in separate 250 mL containers . After cooling to room temperature, the containers were inoculated with plugs of the pathogen. Inoculated cornmeal-sand containers were then incubated at 30 °C for two weeks and shaken vigorously by hand daily to aid rapid colonization. After incubation the colonized cornmeal-sand inoculum was spread over a metal tray to air dry for five days. Once dry, this inoculum was stored in the dark at room temperature for two weeks before being applied in the field. The density of viable microsclerotia within the cornmeal-sand inoculum was enumerated with direct plating of 10–3 to 10–4 serial dilutions onto NP-10 medium70. Ten replicate plates of each dilution were used. The plates were incubated in the dark at 30 °C for 7 days before counting colony forming units (CFU).V. dahliae trial) or not introduced artificially . The control block contained one 20-plant plot per cultivar for a total of 90 plots (90 cultivars). Due to the lack of replication, no comparisons were made between inoculated and control plots at the cultivar level. Nevertheless, control plots were used to assess the overall effects of pathogen presence in the rhizosphere microbiome of the plants.Additionally, in each pathogen trial, one block was established as a non-inoculated control where the soil-borne pathogens were eliminated through chemical fumigation prior to planting  during the growing season and preplant slow release fertilizer banded in the bed for a total input of 112 kg N haDuring fruit set, we determined plant mortality in the 90 cultivars visually by assessing the percentage of dead plants in each plot. Additionally, presence of the pathogens in symptomatic plants was confirmed by plating tissue pieces of internal crown tissue on selective media. Tissue isolations were conducted on 10 symptomatic plants in each replication in the host resistance trial on a biweekly basis. Subsequently, we selected ten representative cultivars exhibiting a range of high to low resistance within each pathogen trial for further study of their rhizosphere microbiome. A summary of the cultivars used in this study with their respective breeding programs is shown in Table 3 bulk soil sample (without the presence of roots) from each plot was also collected from the center of each strawberry bed (50 cm from the nearest plant) at a depth of 10 cm. Rhizosphere soil was collected by dry sieving roots on sterilized 500 µm sieves. Samples were lyophilized and stored at − 80 °C prior to DNA extraction.At harvest (July 2017), we performed another visual evaluation to determine actual plant mortality per plot at the moment of harvest (% of dead plants). Plants were classified as susceptible , moderate , and resistant . One shoot, soil and rhizosphere composite sample was collected subsequently from each plot for each of the selected cultivars Fig. . Briefly72 acid digestion method in the Ultima 2 ICP-OES .Tissue analysis was performed using leaf and petiole samples dried for 24 h at 55 °C. Dry samples were crushed using a mortar and pestle, followed by two 2-min cycles of grinding in a Bel-Art Micro-Mill grinder . Tissue carbon and nitrogen concentrations were analyzed from a 300 mg subsample using a vario MAX CNS Macro Elemental Analyzer . Quality control was ensured by analyzing duplicate samples and NIST SRM 1573A tomato leaf standards every ten samples. Concentrations of leaf P, K, Ca and Mg were determined using a modified USEPA73. PCR reagents included 25 µL of DDH20, 2.5 µL of each primer, 12.5 µL Phusion Hot start flex 2 × Master Mix (Thermo Fisher Scientific), and 50 ng of template DNA. The thermal cycling scheme consisted of 30 s at 98 °C, 10 s at 98 °C, 35 cycles of 54 °C for 30 s, 45 s at 72 °C and 10 min at 72 °C. After the first round of PCR, sequencing adapters and barcodes were added for further amplification. PCR amplification products were visualized by 2% agarose gel electrophoresis. The target fragments were recovered using the AxyPrep PCR Cleanup Kit  according to manufacturer’s instructions. The PCR product was further purified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific). The library was quantified on the QuantiFluor fluorescence quantification system . Each qualified sequencing library  was diluted and pooled/multiplexed. The pooled library was loaded on Illumina MiSeq and the MiSeq Reagent Kit v2, 500 cycles (Illumina) was used for paired-end sequencing (2 × 250 bp). Raw sequence data has been deposited in GenBank (accession number PRJNA666409).To study bacterial and archaeal diversity in the soil and rhizosphere samples, DNA was extracted from a 250 mg subsample using the DNeasy PowerSoil Kit  following the manufacturer’s recommendations. DNA yield was measured on a NanoDrop 2000 . Samples with yield below 5 ng/µL were extracted again prior to amplification. The V4 region of the 16S rRNA was subsequently sequenced using bacterial/archaeal universal primers 515F (5′ -GTG YCA GCM GCC GCG GTA A- 3′) and 806R (3′ -GGA CTA CNV GGG TWT CTA AT-3′)www.drive5.com/usearch). Paired end reads were merged with an allowance of 10 bp max differences and with a maximum allowance of 10 bp differences and > 80% identification. Sequences with less than 50 bp were filtered and all sequences trimmed to 280 bp. Low quality sequences were filtered using a maximum expected error threshold of 1.0. Sequences were clustered into denovo Operational Taxonomic Units (OTUs) based on 97% similarity and singletons were discarded. Taxonomy was assigned to representative OTU sequences using the Naïve Bayesian classifier74 based on the Ribosomal Database Project75. Chloroplast and unassigned sequences were removed from OTU tables prior to statistical analyses. Samples were rarefied to match the sequences of the lowest count sequence of 6,666 for analyses using taxonomic rank abundance.Paired-end sequences obtained on the MiSeq platform were de-multiplexed, followed by raw data filtering and processing utilizing the UPARSE pipeline  ordinations based on the resemblance matrices using a maximum of 50 restarts and the lowest stress solutions. The differences between sample types (rhizosphere or bulk soil), pathogen trials (V. dahliae or M. phaseolina), plant genotypes and resistance to the soil-borne diseases in the soil microbial community composition within each pathogen trial was assessed through analysis of similarities (ANOSIMS) or PERMANOVA  using Spearman rank correlation with 999 max permutations of the resemblance matrices were used to determine significance levels between groups. Similarity percentage (SIMPER) analyses were conducted using the Bray–Curtis similarity matrix. Correlations between rhizosphere OTU relative abundances and plant variables  were made using the distance-based linear model (DistLM) with 999 permutations. All microbial community data were analyzed using PRIMER-e version 7 with the PERMANOVA + addon .Resemblance matrices for the OTU tables were generated using Bray–Curtis similarity of the standardized and Log (x + 1) transformed datawww.huttenhower.sph.harvard.edu/galaxy) with an LDA threshold of 2.0 and an alpha value of 0.05.The effect of ‘sample type’ (bulk vs. rhizosphere soil), ‘cultivar’ and ‘cultivar resistance’ to the fungal pathogens on the microbial diversity (Shannon diversity index), plant mortality, and plant nutrient comparisons among groups were conducted using general linear models and Tukey HSD post hoc tests in JMP Pro ; Pearson’s correlations were carried out among the different plant traits analyzed using JMP Pro 13. A list of bacterial genera known to include species antagonistic to filamentous fungal plant pathogens was used to compare treatment groups . Linear discriminant analysis (LDA) effect size (LEfSe) comparing taxonomic features between groups was conducted online (Supplementary Information.


Coffee is a widely consumed beverage, both in Europe, where its consumption is highest, and on other continents. It provides many compounds, including phenolic compounds. The aim of the study was to assess the effect of various brewing methods on the total phenolic content (TPC) in the infusion. Research material comprised commercially available coffees: Instant Arabica and Robusta, freshly ground Arabica and Robusta , ground Arabica and Robusta, decaffeinated Arabica, and green Arabica and Robusta. The following preparation methods were used: Pouring hot water over coffee grounds or instant coffee, preparing coffee in a percolator and using a coffee machine. Additional variables which were employed were water temperature (90 or 100 °C) and its type (filtered or unfiltered). In order to determine the impact of examined factors, 225 infusion were prepared. Total phenolic content was determined by the spectrophotometric method using the Folin-Ciocalteu reagent and the obtained results were expressed in mg gallic acid (GAE) per 100 g of brewed coffee. The highest value was obtained for 100% Arabica ground coffee prepared in a coffee percolator using unfiltered water at a temperature of 100 °C: 657.3 ± 23 mg GAE/100 g of infusion. High values were also observed for infusions prepared in a coffee machine, where the highest TPC value was 363.8 ± 28 mg GAE/100 g for ground Arabica. In turn, the lowest TPC was obtained for Arabica green coffee in opaque packaging, brewed with filtered water at a temperature of 100 °C: 19.5 ± 1 mg GAE/100 g of infusion. No significant effect of temperature and water type on the TPC within one type of coffee was observed. Due to its high content of phenolic compounds, Arabica coffee brewed in a coffee percolator should be the most popular choice for coffee drinkers. Coffee is a widely consumed beverage, drunk primarily for its stimulating effect and aroma. Due to its worldwide popularity, it occupies an important place in international trade. The 2018 data show that the total production of coffee beans exceeded 10 million tons, an increase of 4.6% compared to the previous year. The leading producer of coffee is Brazil, where 3.8 million tons of beans were produced in 2018   ,20,21.In order to estimate the daily consumption of phenolic compounds with coffee, the infusion with the highest TPC value was selected for each type of coffee. The value was multiplied by four cups (average coffee consumption per day) .The results of analyses of the total content of phenolic compounds depending on the method of brewing in various types of coffee were statistically analyzed using Statistica 13.3 and Excel.p < 0.05. Additionally, descriptive statistics were determined: arithmetic means, standard deviations, minimum and maximum values, medians as well as lower (Q1) and upper (Q3) quartiles and the range between them (IQR). Additionally, Principal Components Analysis (PCA) was performed to reduce data and identify main factors responsible for variability.The normality of the data distribution was verified by appropriate tests: Shapiro-Wilk, Kołmogorow-Smirnow and Lilliefors. The Wilcoxon sequence test was used to assess differences in TPC values between individual coffee brews. The level of significance in the analyses was Brewing methods—using a coffee machine, a coffee percolator and pouring water from a kettle—have a significant impact on the total phenolic content in a coffee brew, which was confirmed by the PCA analysis. The most recommended method for all ground and freshly ground coffee is a coffee percolator. TPC values for instant coffee infusions were higher in comparison to those for ground and freshly ground coffee brewed by pouring water over coffee grounds. The infusion prepared from green coffee was characterized by a lower TPC value than that from roasted coffee, while higher TPC values were observed for coffee in transparent packaging in comparison to opaque packaging. Water temperature and type of water used had no effect on TPC.