The mechanisms demonstrated here for terrestrial impact ejecta modify our understanding of the effects of impact processing on the isotopic evolution of planetary crusts.Fractionation effects related to evaporation and condensation had a major impact on the current elemental and isotopic composition of the Solar System. Although isotopic fractionation of moderately volatile elements has been observed in tektites due to impact heating, the exact nature of the processes taking place during hypervelocity impacts remains poorly understood. By studying Fe in microtektites, here we show that impact events do not simply lead to melting, melt expulsion and evaporation, but involve a convoluted sequence of processes including condensation, variable degrees of mixing between isotopically distinct reservoirs and ablative evaporation during atmospheric re-entry. Hypervelocity impacts can as such not only generate isotopically heavy, but also isotopically light ejecta, with δ Fe isotopic composition of the distal ejecta of a terrestrial impact crater records both evaporation and condensation, refining the nature of the isotopic fractionation taking place during hypervelocity impacts in the Solar System. Collision was an important source of heat in the early Solar System potentially also leading to evaporation, in conjunction with radioactive decay and gravitational heating2. The isotope ratios of the moderately volatile elements are sensitive to evaporation and condensation. Altered isotopic compositions of these elements are observed for the Moon3, formed as the result of a giant impact, for the volatile-depleted parent asteroids of the ureilite4 and angrite5 meteorites, for asteroid Vesta6, and potentially also for Mercury with its partially stripped mantle7.Evaporation and condensation are fundamental processes in the chemical evolution of the Solar System. When the protosolar dust cloud evolved into the rocky terrestrial planets, the asteroids and planetesimals accreting to these planets were significantly depleted in moderately volatile elements as the result of these processes8. Microtektites are the sub-millimeter analogs of tektites, thought to have solidified from smaller droplets of melt. Both tektites and microtektites are ejected from the source crater, travel ballistically, are quenched in flight, and are deposited hundreds to thousands of kilometers away from the source crater, in so-called strewn fields. A key feature of tektites is their volatile-depletion, observed for water, as well as for volatile and moderately volatile elements9.Terrestrial ejecta, which formed during hypervelocity impacts that were especially prevalent during the early Solar System, are particularly interesting for studying the effects of evaporation that occurred during these events. Such impacts generate ejected silicate melt with broadly upper crustal compositions as a result of extreme heat and pressure. Tektites are distal impact glasses formed by melting of terrestrial rocks upon hypervelocity meteorite impacts10, as well as the occurrence of high-pressure mineral phases in some tektites11. The recovery of shock metamorphosed rock and unmelted mineral fragments in Australasian microtektite layers in the South China Sea12 further supports the impact origin. Australasian microtektites have been collected from deep-sea sediment cores from the Ocean Drilling Program (ODP), Deep Sea Drilling Project (DSDP), as well as from piston cores by a variety of oceanographic institutes in over 60 locations in the Indian and western Pacific Oceans and adjacent seas13, and from several sediment accumulation traps in Antarctica17.Of the four historically recognized strewn fields, the Australasian tektite and microtektite strewn field is the largest and most recent example. Australasian tektites and microtektites are linked to a hypervelocity impact origin by their geochemical compositions and features characteristic for shock melting: the absence of primary minerals, the presence of lechatelierite and shocked mineral grains in a subset of tektites and microtektites56/54Fe = 0.00 ± 0.22‰, 2SD) is close to the Solar System average 18. The isotopic composition of Fe in the upper continental crust, as estimated from the composition of loess-paleosol sequences19, is slightly heavier . Terrestrial basalts have δ56/54Fe of 0.08 ± 0.13‰ (2SD), while differentiated SiO2 rich rocks display heavier Fe isotopic values in the range of 0.1–0.5‰18. Iron is a comparatively refractory element . Thus, energetic events are required for evaporation to play a significant role in the Fe isotope budgets of planetary bodies. Since early reports on Fe isotopic compositions of the rocky bodies in the inner Solar System, it has been suggested that the observed differences between the Moon, Earth, Mars, and some asteroids may result from variable degrees of evaporation experienced during their evolution22. Although since then, it has become clear that the Fe isotopic composition of planetary surfaces is affected by both core-mantle segregation24 and redox-dependent25 mantle differentiation27, these processes alone may not be sufficient to account for the observed signatures of meteorites28. As such, evaporation remains a viable process that could affect the Fe isotope budgets during planetary evolution either at the nebular stage, during the accretion, or subsequently following episodes of hypervelocity impacts or magmatism29. Fractionation of Fe isotopes in impact plumes was observed earlier in the case of metal grains of CB chondrites30. The process of thermal evaporation of a target rock is typically associated with a loss of volatile components, shifting the residual melt towards heavier isotopic compositions.Iron is a major rock-forming element in most planetary reservoirs with variable geochemical behavior in an assortment of contexts. Iron isotope ratios have been used as a key tracer of high-temperature Solar System processes, but also as a useful tool to study the geochemical cycle of Fe in low-temperature environments. The Fe isotopic composition of Earth’s mantle rocks , which correlate with the extent of element depletion as a result of evaporative loss36. The Cd isotopic composition of a single tektite from Laos37 displayed an isotopic composition 0.76‰ per amu heavier than the average terrestrial composition. At the same time, other volatile elements exhibit no isotope fractionation due to evaporation in macroscopic tektites. As an example, the isotopic variations of Li and K only reflect mixing of distinct reservoirs within an inhomogeneous target40.Isotope fractionation due to partial distillation during impact processes has a direct bearing on our understanding of key processes in the planetary sciences, such as asteroid or planetary collisions, for example during accretion or the Moon-forming impact41. The observation of extreme K isotope fractionation in microtektites, with no significant variations in their macroscopic analogs34, implies that microtektites may be more sensitive to evaporation in hypervelocity impact processes.Due to their sub-millimeter size, microtektites have different surface-to-volume ratios and heating histories as a function of time, as compared to macroscopic tektites. While macroscopic tektites were studied intensely for potential evaporative isotope fractionation, only a single study so far has focused on microtektites, reporting K isotope ratio data for 28 Australasian microtektitesThe main aim of this work is to assess and refine the mechanisms of isotope fractionation taking place during hypervelocity impact events and ejecta emplacement by studying the Fe isotopic compositions of Australasian microtektites. To determine the Fe isotopic compositions of these microscopic glasses in situ, we use a laser ablation unit coupled to a multicollector ICP mass spectrometer. The mechanisms of evaporation during impact events currently remain poorly understood. Thus, the composition of the microscopic ejecta particles formed in a single impact event 790 ka ago may improve our understanding of the effects of collisions on the isotopic evolution of planetary crusts, and may ultimately improve the use of Fe as a proxy of planetary processes.Here, we study a collection of 48 microtektites from 14 locations in the Indian and Pacific Oceans and Antarctica, and 13 macroscopic tektites from Thailand, Vietnam, the Philippines, Indonesia and Australia, distributed across the Australasian strewn field Fig. .Fig. 1Co12 was used here to calculate great circle distances, although other suggested locations42 would provide nearly equal distances. Smaller microtektites tend to display isotopically lighter Fe isotopic signatures . A frequency distribution of the Fe isotope ratio variation is presented in Fig. res Fig. . Microte35. The results of this work show no significant variation in the Fe isotopic composition of macroscopic tektites (δ56/54Fe between −0.30 and +0.14‰), suggesting that evaporation did not play a significant role in the evolution of their Fe isotopic signatures. The δ56/54Fe values and FeO concentrations in microtektites, found in ODP sediment cores drilled in the South China and the Sulu Seas indicate only marginally significant Fe isotope ratio variability. The δ56/54Fe values cluster near average continental crustal values from −0.46 to +0.29‰. The measured data for the microtektites from these sites are also not consistent with the trends expected for evaporative fractionation. Relative to the other microtektites analyzed in this work, the microtektites from the South China and the Sulu Seas were deposited closer to the hypothetical impact crater location (1300–2350 km12). Their trace element signatures indicate minimal, if any, evaporation following their residence in the impact plume and ballistic transfer , f equal to 0.71 – 0.58 and a fractionation factor α between 0.995 and 0.997. However, the Fe isotopic signatures of Australasian microtektites do not simply depend on the distance from the crater  in macroscopic tektites, without additional details on the exact mechanism10. Particles originating from hotter zones in the impact plume experienced larger degrees of evaporation and have at the same time been accelerated to higher velocities, leading to their deposition farther from the impact crater. This evaporation mechanism, previously demonstrated for a collection of Australasian microtektites and for microtektites from the Larkman Nunatak17, in which the abundances of the volatile alkali elements decrease progressively with the flight distance45, appears to be similarly applicable to the Fe isotope fractionation observed in this work.The distinct Fe isotopic signatures of microtektites between more proximal (light) and distal (heavy) collection sites, with Antarctic microtektites having experienced the largest evaporation, indicates Fe isotope fractionation in the hotter and cooler areas of the impact plume. It has previously been shown that impact glass particles that form during the earliest stages of the impact cratering process, in the hottest and fastest part of the impact plume closer to the growing transient cavity, undergo more evaporation and homogenization, are smaller, and contain less vesicles and mineral inclusions46, gas drag cannot contribute to heating of the microtektites over most of their ballistic trajectories, as they escape the atmosphere. However, our model based on standard equations for a meteoroid entry with drag and ablation  indicates that the most remote Australasian microtektites, which reach Antarctica, must have experienced velocities and trajectory angles that could lead to partial vaporization and mass loss during atmospheric re-entry. As shown in Fig. 47.While the extreme conditions of hypervelocity impact events are likely the primary factor controlling the evaporation and isotope fractionation in microtektites, ablative evaporation during flight and interaction with the atmosphere may also take place. Microtektites have cooled quickly after pressure release and plume expansion, so by the time they reach space, the evaporation ceases. Different from the much faster cosmic spherules56/54Fe observed for ODP microtektites must be related to a heterogeneously distributed isotopically light phase during the turbulent impact plume stage. Turbulence is an important property inherent to the structure of impact plumes, and thus perfect geochemical trends with distance are not to be expected in general.The light Fe isotopic signatures observed for a fraction of the microtektites however require a different mechanism. Whilst most Antarctic microtektites are fractionated towards heavier Fe isotopic signatures, a small number of microtektites from the Transantarctic Mountains (TAM) and Larkman Nunatak (LN) do not exhibit positively fractionated values. This may suggest that the SRM microtektites underwent slightly different processes during the impact event compared to the Antarctic microtektites from the TAM and LN sites. Alternatively, TAM and LN microtektites could contain a higher proportion of an isotopically light Fe component, a product of condensation, relative to those from the SRM. However, the major and trace element geochemistry of these groups does not record such processes or otherwise these processes led to similar concentrations. Similarly, the variation of nearly ~1‰ in δ56/54Fe value of −2.85‰, observed for microtektite 137-8 from the V19-153 location in the Eastern Indian Ocean, cannot be explained based on a continuously evaporating melted reservoir. These light signatures are also unlikely to represent initial isotopic heterogeneity within the target material48. Most terrestrial (igneous) rocks are characterized by δ56/54Fe values close to zero18. Certain sulfides49 and carbonates51 display light Fe isotopic compositions, but a sulfide- or carbonate-rich target cannot explain the Fe signatures of microtektites based on the bulk geochemistry and Fe isotopic signatures of macroscopic tektites. The Australasian microtektites studied here are highly pristine, and no weathering features, which could alter the Fe isotopic signatures, are observed. The major and trace element concentrations measured earlier and in this work45 confirm that limited to no alteration affected the microtektites , both in positive and negative direction, which is in full agreement with the Fe isotope fractionation observed here. The observation that microtektites closer to the source location contain slightly less K relative to macroscopic tektites is consistent with the incorporation of a condensed component in Australasian microtektites45. In the case of macroscopic tektites, the effect of condensation is likely insignificant as a result of their low surface-to-volume ratios53, and light isotopic signatures of Fe and K have not been observed to date.Thus, the extremely light Fe isotopic signatures of microtektites down to δThese observations provide strong evidence that condensation during the impact event played an important role in the formation of at least a fraction of the ejecta produced. Moreover, microtektites indicate overprinting of heavy Fe isotopic signatures by light vapor condensate.31 or mixing with an isotopically light component in the impact plume. Evaporation is not restricted to Antarctic microtektites only, as one microtektite from ODP 769A, another particle from ODP 1144A, and a third one from V20-138 also display slightly positive δ56/54Fe values. However, most microtektites from the Indian and Pacific Oceans demonstrate signatures approximating zero or display negative δ56/54Fe values, suggesting more complex fractionation histories. The microtektites from the V20-138 and V19-153 locations in the Pacific and Indian Ocean, respectively, both show weakly negatively correlated δ56/54Fe and FeO contents. The Fe isotopic compositions for each of these locations can loosely be explained by Rayleigh distillation expected for thermal evaporation. However, compared to the putative target composition, these trends suggest that the microtektites from V20-138 and V19-153 formed by evaporation from the material that initially had an extremely light Fe isotopic signature (δ56/54Fe between −2.61 and −2.85‰) and an elevated FeO content between 7.19 and 7.32 wt%. Such initial compositions are in agreement with the lightest isotopic signatures preserved among the microtektites measured here, as a result of condensation. If only evaporation was responsible for these signatures, this would imply fractionation factors (α) equal to 0.998–0.9935 and up to 80% FeO loss due to evaporation. In addition, these microtektites indicate stronger scatter along the Rayleigh curve. Such signatures are thus best explained by a combination of (i) isotope fractionation of the melt towards heavier values due to evaporation and (ii) mixing with variable amounts of a re-condensed, isotopically light component.The relation between the Fe isotopic signatures and FeO contents of the Australasian microtektites, including those from Antarctica, implies more convoluted processes driving their Fe isotope fractionation beyond single Rayleigh-style evaporation10, the formation of all microtektites solely by condensation is not possible.However, the mixed Fe isotopic signatures cannot be explained by assuming that all microtektites form within the plume as fractional condensates that subsequently underwent partial evaporation during atmospheric re-entry in the case of sufficiently high velocities. Based on the presence of abundant vesicles and the occurrence of mineral inclusions that display some indications of melting in particular specimens54. In this case, the evaporation and condensation regimes must coexist in the rapidly expanding impact plume. Within the first few seconds after the impact, molten, shocked and partially vaporized materials from the upper target layers are ejected. The resulting impact plume is a turbulent mixture of vapor, melt, and solid fragments with wide compositional and thermal ranges. The temperature of the ejected materials is mainly defined by the melt-vapor transition of quartz and does not exceed ~4000 K. As the plume expands, the temperature and pressure of the ejected material start to drop quickly along the pressure release adiabat on the phase diagram  and cools down faster than the central part of the plume, which is characterized by lower velocities and a higher density. In this context, the cooling rates are known to be an important factor affecting the final isotopic composition of the condensed material31.Although evaporation during ballistic transport and interaction with the atmosphere cannot be excluded in the case of the most distally deposited (Antarctic) microtektites, evaporation must also take place earlier on in the impact plume, as suggested for lunar glass beads that form in the absence of an atmosphereram Fig. . Dependiram Fig. . The par55. Similarly, a thin layer of material covering the inner part has been observed for lunar glasses, suggesting heterogeneous condensation in volcanic56 and impact57 plumes. As no isotopically light microscopic glass58 was found in the vicinity of the crater, homogeneous condensation may not have been a prevailing mechanism in the plume. Because the impact event produces relatively little material58 with extremely high compression >300 GPa  and FeO (0.986) species, suggesting a more complex mechanism. This implies non-equal evaporation coefficients γ for the Fe isotopes in microtektites, in agreement with a diffusion-limited evaporation regime, as proposed for Cu and Zn in macroscopic tektites59. Alternatively, a model of evaporation from a supercritical fluid during rapid cooling may be invoked, the so-called “bubble-stripping” evaporation mode, which is suppressed at high pressures60. In the past, local partial re-condensation in the thin layer above the surface of the microtektite during evaporation was used to explain non-ideal Rayleigh distillation32. However, due to their supersonic velocity, microtektites are expected to lose such vaporized shell before it re-condenses. The mean free path of an evaporated species in the vapor is likely to be large compared to the particle size, making it unlikely for these species to re-condense. Finally, the deviations of the fractionation factors found to best fit the microtektite data from predictions of ideal evaporation to vacuum may be explained in terms of the Hertz–Knudsen theory by Fe partial pressures that are 64 to 93% of the saturation pressure during evaporation31. As ideal Rayleigh fractionation factors α are rarely achieved in natural systems35, the range of fractionation factors (α = 0.995–0.999) fitted for Fe isotope fractionation in microtektites may be used as a first order estimate to interpret Fe isotope fractionation in terrestrial hypervelocity impact events.The Fe isotopic signatures of microtektites suggest Rayleigh evaporation under non-ideal conditions. Laboratory vacuum evaporation of FeO has previously constrained the ideal fractionation factor α, while evaporation of mixtures of oxides resulted in slightly lower fractionation magnitudes than expected for the Rayleigh process32 are often used to normalize the content of the element fractionated in the evaporation process. In the case of the microtektites, such normalization does not simplify the observed patterns, confirming a more convoluted process , and their related counterpart, high-Al, Si-poor precipitates (HASP) formed within impact plumes on the Moon as condensates and partial evaporates, respectively54, and are found as glassy microscopic spheroids in lunar regolith breccias. Evaporate and condensate glasses with structures and compositions similar to GASP and HASP have also been observed in terrestrial zhamanshinite impact glass64. Similar signatures of condensation should be searched for in Martian regolith breccia65. Although no Fe isotope fractionation due to condensation was observed in bulk lunar breccia61, GASP and HASP phases are likely to be characterized by strongly fractionated isotope ratios for a range of elements based on their geochemical compositions66. Similarly to microtektites, light isotopic signatures for Fe, Cu, Zn, and Cd in lunar orange glass beads68 imply condensation in volcanic plumes during pyroclastic eruptions on the Moon.The observations made here have important implications for specific Solar System processes. Evaporation and condensation in the early Solar Nebula are known to play a significant role in the Fe isotope budgets of chondrules, which experienced an episode of evaporation and re-condensation and have a δ4. Although the average Fe isotopic composition of the microtektites approximates that of the Bulk Silicate Earth, with a near-Gaussian frequency distribution  and ODP 1144 A cores (particles 35-38), as well as the Antarctic microtektites from TAM  were provided by LF45. The Antarctic microtektites from LN are from the collection of MG17. Sample collections in Antarctica were organized in compliance with the Antarctic treaty. The microtektites collected from the sea sediments V19-153 (137-7 to 137-12), V16-76 (137-5 and 137-6), RC9-143 , V20-138 (197-1 to 197-6 and 463-3 to 463-6) and V28-238 (188-3), as well as the microtektites from the ODP 768B core , are part of the collection of BG69. Several of these microtektites were characterized in terms of their petrology and geochemistry in previous studies69. The macroscopic tektites are collected within the Australasian strewn field in Philippines , Indonesia , Thailand , Vietnam (Saigon BT-2), and Australia (MA-142)44. The macroscopic Javanites JAV1.1, JAV1.5 JAV1.8, JAV0.9, Saigon BT-2 and a single Philippinite are from the VUB collection44. The macroscopic Philippinite PMJ-20 and MA-142 Australite flanged button were purchased from a commercial vendor. The Muong Nong tektites FT-598 and FT-118, tumbled Indochinites TI8A and TI8B and the Javanite JAV-32 are from the collection of Darryl Futrell. The latitudes and longitudes for the collection sites are presented in the ESI.The SRM#1-7 Antarctic microtektites from Sør Rondane Mountains, Widerøefjellet, were collected by SG and characterized by BS71. In situ high-precision Fe isotope ratios in geosciences are often measured on SIMS72. Although SIMS has somewhat superior spatial resolution and its isotope ratio measurement precision is nearly similar to that of LA-MC-ICP-MS, the latter is less prone to matrix and topography effects and has more flexibility for instrumental mass discrimination correction. In this work, Fe isotope ratios of the Australasian macroscopic tektites and microtektites were measured at A&MS-UGent by using a setup consisting of a Teledyne CETAC Technologies Analyte G2 laser ablation system, based on a 193 nm ArF*excimer laser with a pulse duration <5 ns and equipped with a HelexII double-volume ablation cell, coupled to a Thermo Scientific Neptune MC-ICP-MS unit, equipped with a large volume jet interface pump and high-efficiency jet interface. The instrumental mass discrimination was corrected for by a combination of internal standardization using a co-nebulized Ni standard solution and external correction using USGS BHVO-2G or BCR-2G glass reference materials as external standards measured in a sample-standard bracketing sequence. MPI-DING glass reference materials ML3B-G, ATHO-G, GOR128-G, and StHS6/80-G were used for daily validation of isotope ratio accuracy and precision. The analysis was performed by ablating along a line with a laser spot size of 10–30 µm and a laser repetition rate of 10–30 Hz. Validation of the protocol was performed using MPI-DING and USGS glass reference materials and cosmic spherules, which have highly variable matrices and Fe contents. The results of LA-MC-ICP-MS were in full agreement with the Fe isotopic signatures found in literature and with the results obtained following acid digestion, chromatographic Fe isolation and MC-ICP-MS measurement, independent of matrix compositions and the magnitude of the Fe signatures. LA-MC-ICP-MS δ56/54Fe values of the glass GRMs and cosmic spherules, when plotted versus their δ56/54Fe values measured after acid digestion, fall on a 1:1 line (0.9561 ± 0.0080) with MSWD = 1.746. For the in-length description of the protocol and the validation of the method, the reader is referred to previous work46.LA-MC-ICP-MS has proven valuable to provide high-precision Fe isotope ratio data in the geosciences, when either spatially resolved information is needed, or when the size of the target restricts its analysis by a conventional solution approach2 to minimize spectral interferences otherwise hampering accurate measurements of elements in the low mass range. Quantification of the major elements was performed relying on external calibration against MPI-DING and USGS glass reference materials combined with normalization of cumulative element oxide level to 100%73. The heavier trace element compositions were measured in a separate LA-ICP-MS run with the collision/reaction cell operated in vented mode  and by focusing the laser beam to a larger spot with a diameter of 80 µm in order to achieve higher signal intensities. The trace elements were quantified relying on external calibration against MPI-DING and USGS glass reference materials. One of the major elements (Cr or Mn), quantified in a previous run, was used as internal standard. Such approach allows for a solid sample analysis without preliminary knowledge of the concentration of an internal standard.Concentrations of the major elements in microtektites were measured at A&MS-UGent by using a setup consisting of the Analyte G2 laser ablation system coupled to an Agilent Technologies 8800 tandem ICP-MS instrument. The mass spectrometer was operated in MS/MS mode and the collision/reaction cell was pressurized with H56/54Femelt = (δ56/54Fe0 + 1000) f(α−1) − 1000 and δ56/54Fevapor = (δ56/54Fe0 + 1000) αf(α-1) − 1000, where the delta notation reflects the relative difference between the 56Fe/54Fe isotope ratios of the melt, vapor and the initial phase from that of an internationally accepted IRMM014 standard in per mil: δ56/54Fe = ((56Fe/54Fe)/(56Fe/54Fe)IRMM014 − 1)·1000. f is the fraction of the 54Fe evaporated and α is a fractionation factor that scales to the square root of the masses32. The fractionation factor α between the residue and the gas in the case of evaporation to vacuum is defined by the Hertz–Knudsen equation α = (γ56/γ54)(m56/m54)0.5, where m represents the masses of the Fe isotopes and γ the corresponding evaporation coefficients. At first estimation, the ratio of the evaporation coefficients for different isotopes of the same element approximates unity. The delta notations for Cu, Zn, and Sn discussed in the text are calculated in a similar way as for Fe using the 63Cu, 64Zn, and 118Sn reference nuclides.In the process of evaporation, the isotopic compositions of the remaining melt and instant gaseous phase are calculated using the Rayleigh fractionation equation: δ74 and includes phase transitions. Radiative energy transfer and kinetics of vaporization/condensation are not included. Shock physics code SOVA75 was used to model the early stage of impact cratering and ejection of potential tektites and microtektites. The code is coupled with the ANEOS76 package for quartz target material74 and a dunitic projectile. A projectile 2 km in diameter with an impact velocity of 22 km s−1 at 45o to the horizon and porous material at the upper 100 m of the target (sand) were used to model the Australasian impact, which results in a diameter of the transient cavity of about 18–22 km. The initial stage modeled in this work can easily be scaled to any crater size. Moreover, ejection velocities and shock pressures do not depend on the projectile size. Tracer particles are used to track the ejection of tektites.The impact event is modeled with 25 m resolution (50 × 50 × 50 m cell), the equation of state is based on pure quartz78. The drag equation was modified for a molecular flow regime:a is density of the atmosphere. The incoming energy Ein = 0.5ρaSV3 is balanced by radiation and vaporization. Assuming that particles are small enough to be isothermal with temperature T and emissivity ε, the radiative energy is Erad = 4 SσεT4, where the Stefan-Bolzmann constant σ = 5.67 × 10−8 W/m2/K4. Evaporation into a vacuum can be described by the Langmuir equation:C = 4.377 × 10−3, µ is molecular weight, Pv is vapor pressure provided by the Antoine equation Pv = A−B/T. Finally, temperature was calculated from a non-linear equation:79. However, such simplification leads to a substantial temperature overestimate. Although there is no obvious dependence for temperature on the particle size, the particle velocity dependence on altitude  is a function of the particle size. An emissivity of 0.95 is used in all simulations. Atmospheric density is defined by the ATMCIRA data. A list of constants used for the numeric modeling is provided in Table A standard set of differential equations was used to model re-entry of high-velocity particles into the atmosphereSupplementary InformationPeer Review FileDescription of Additional Supplementary Files


In bladder cancer, up to 70% of patients will relapse after resection within 5 years, in which the mechanism underlying the recurrence remains largely unclear.t-test, χ2 test, Kaplan Meier and Cox regression analyses, and Spearman's rank correlation coefficients were used for statistical analyses.Quantitative real-time PCR, western blot and immunohistochemistry were conducted. The assays of tumor sphere formation and tumor xenograft were further performed to assess the potential biological roles of ATF5 (activating transcription factor 5). Chromatin immunoprecipitation-qPCR and luciferase activity assays were carried out to explore the potential molecular mechanism. A two-tailed paired Student's DVL1 gene, resulting in activation of Wnt/β-catenin pathway.ATF5 is elevated in bladder urothelial cancer (BLCA) tissues, especially in recurrent BLCA, which confers a poor prognosis. Overexpressing ATF5 significantly enhanced, whereas silencing ATF5 inhibited, the capability of tumor sphere formation in bladder cancer cells. Mechanically, ATF5 could directly bind to and stimulate the promoter of This study provides a novel insight into a portion of the mechanism underlying high recurrence potential of BLCA, presenting ATF5 as a prognostic factor or potential therapeutic target for preventing recurrence in BLCA. Globally, bladder cancer is the 10th most prevalent cancer type, with about 549,000 newly diagnosed patients in 2018 −[ as instructed by the manufacturer and used for cDNA synthesis with random primers. Normalize the expression data to Western blot assay was conducted as reported previously . The antIHC was carried out in 140 BLCA tissues, which were detected with an anti-ATF5 antibody  as previously reported . ImmunosATF5 gene from cDNA was done by PCR and later cloned into a pcDNA4 lentiviral vector. For silencing ATF5, cloning of 2 human ATF5-targeting short hairpins RNA (shRNA) sequences into a PLKO.1  was done to enable the generation of corresponding pSUPER. retro. ATF5-RNAi(s). SW780 or UM-UC-3 cells were seeded in culture plates (2 × 106 cells/p100 plate). Transfection was done using 10 ug of designated plasmids. Cells stably expressing ATF5 or ATF5 shRNA were obtained through retroviral infection of the 293FT cells. These cells were treated for 3 days using puromycin (3.33 µg/mL). The promoter region of the human DVL1 gene, which had been generated via PCR-amplification from SW780 cells were cloned into NheI/BglII sites of the pGL3-basic luciferase reporter plasmids  in order to establish DVL1 luciferase reporters. Tables Amplification of the human 2) were plated in 6-well ultralow cluster plates and incubated for 10 to 12 days. Incubation of tumor spheres was done in serum free DMEM/F12  with epidermal growth factor , B27 , basic fibroblast growth factor , insulin , and BSA . After 10–12 days, tumor spheres  were counted and imaged by inverse microscopy. The efficiency of sphere formation was calculated using the formulacolonies/input cells × 100%.Cells (5 × 104 cells per well) were plated in 24-well plates and incubated for 24 h. Transfection of specified plasmids as well as a pRL-TK Renilla plasmid (1.5 ng) was done using the Lipofectamine 3000 Reagent . Then, 48 h after transfection, the luciferase as well as Renilla signals were assessed by the Dual Luciferase Reporter Assay Kit  as instructed by the manufacturer.Luciferase reporter test was conducted as described previously. In short, in triplicates, bladder cancer cells , which had been stably transfected with RNAi-vector, ATF5-RNAi#1, ATF5 or vector, with Matrigel . An external caliper was used to assess tumor volumes, which were determined by the equation (L × W2)/2. Then, 36 d post-inoculation, mice were euthanized by quick intraperitoneal injection of 100 μg/g pentobarbital sodium, and sacrificed for tumor excision.Male BALB/c nude mice  were bought from Guangdong Experimental Animal Center  and housed in an animal holding facility on a 12 h dark/light cycle. Randomly, mice were allocated in to four groups (n = 6 per group). Inguinal folds of mice were inoculated with SW780 cells (1 × 10http://software.broadinstitute.org/gsea/msigdb/The BLCA database from The Cancer Genome Atlas (TCGA) was analyzed by Gene set enrichment analysis  software program (version 2.2.3).t-test. The association between ATF5 levels and clinic-pathological characteristics was calculated by χ2 test. Kaplan Meier was used for establishment of survival curves, which were compared by log-rank test. Then, survival data were calculated by univariate as well as multivariate Cox regression analyses. The bivariate correlation between variables was evaluated by Spearman's rank correlation coefficients. The P-value < 0.05 was the threshold for significance.The SPSS v.21.0 software  was used for all analyses. Comparison of means between groups was done by a two-tailed paired Student’s ATF5 mRNA expression was preliminary analyzed between BLCA tissues and paratumor tissues in an available BLCA data from The Cancer Genome Atlas (TCGA) database. Expression levels of the ATF5 gene in BLCA were markedly elevated than those of adjacent normal bladder tissues , stage (P = 0.014), tumor multiplicity (P = 0.004), recurrence (P = 0.022) as well as progression (P = 0.016), however, they were not correlated with gender, tumor size or age  were stained using a human ATF5 antibody. ATF5 expression in BLCA specimens was markedly elevated than in paratumor tissues , DVL1 protein  and the active-β-catenin expression  in eight fresh BLCA samples  of ATF5 and DVL1 in TCGA-BLCA datasets were shown in Fig. ATF5 and DVL1 mRNA levels were tightly related to the CNV (Fig. ATF5 and DVL1 predicted a higher gene expression, while the deletion in CNV of AFT5 and DVL1 predicted a lower gene expression (Fig. ATF5 was not directly associated with DVL1 gene expression (Fig. ATF5 CNV does not contribute to the ectopic expression of DVL1 gene. Although aberrant CNV of DLV1 contributed to ectopic expression of DVL1 gene, differences in survival outcomes between DVL1 amplification or deletion patients were insignificant (P = 0.379, Fig. ATF5 amplification was found to be a predictor of worse survival outcomes than those without ATF5 amplification (P = 0.014, FATF5 amplification plays a role in ATF5 overexpression in bladder cancer, which conferred a poor prognosis.In various cancers, the CNV Fig. C and D. ion Fig. E and F. ion Fig. G, indicaThis study revealed that ATF5 facilitates the formation of tumor sphere, promotes tumorigenicity and stimulates the Wnt/β-catenin pathway in bladder cancer. Furthermore, ATF5 was upregulated in human BLCA and elevated ATF5 was related to relapse-free survival outcomes, implying that ATF5 might be a potential prognosis marker for BLCA recurrence.ATF5 has been demonstrated to be highly expressed in undifferentiated neural progenitor/stem cells –19. It sDVL1, leading to the stimulation of Wnt/β-catenin signaling.The Wnt/β-catenin signaling, known as the canonical Wnt pathway, is essential for development of the embryo as well as tissue self-renewal of tissues , 36. AbnDVL1 promoter and stimulate its expression, and then activate the downstream genes of the Wnt/β-catenin pathway, including active β-catenin, MYC, CD44, JUN as well as CCND1, whereas down-regulating ATF5 reduced the expression of DVL1 and these factors. These findings demonstrate a novel mechanism underpinning hyperactivation of the Wnt/β-catenin pathway in BLCA. Herein, this study indicates that ATF5 could simulate the Wnt/β-catenin pathway and promote tumorigenic capability.DVL1, as a main component of the Wnt pathway, takes part in transduction of Wnt signals to β-catenin, and then stimulates downstream effector factors . In thisDVL1 expression and stimulates the Wnt/β-catenin signaling, therefore increasing tumorigenicity, enhancing a TIC-like phenotype as well as predicting poor survival. Evaluation of the role of ATF5 in BLCA will broaden our understanding of the mechanism underpinning the high recurrence rate of BLCA, and establish whether ATF5 serves as a prognosis marker or potential treatment target for BLCA recurrence.The present study reveals that overexpression of ATF5 in BLCA directly promotes


Physical condition is important for the ability to resist various parasites and diseases as well as in escaping predators thus contributing to reproductive success, over-winter survival and possible declines in wildlife populations. However, in-depth research on trends in body condition is rare because decades-long datasets are not available for a majority of species. We analysed the long-term dataset of offspring covering 34 years, male parents (40 years) and female parents (42 years) to find out whether the decline of Tengmalm’s owl population in western Finland is attributable to either decreased adult and/or juvenile body condition in interaction with changing weather conditions and density estimates of main foods. We found that body condition of parent owl males and females declined throughout the 40-year study period whereas the body condition of owlets at the fledging stage very slightly increased. The body condition of parent owls increased with augmenting depth of snow cover in late winter (January to March), and that of offspring improved with increasing precipitation in late spring (May to June). We conclude that the decreasing trend of body condition of parent owl males and females is important factor probably inducing reduced adult survival and reduced reproduction success thus contributing to the long-term decline of the Tengmalm’s owl study population. The very slightly increasing trend of body condition of offspring is obviously not able to compensate the overall decline of Tengmalm’s owl population, because the number of offspring in turn simultaneously decreased considerably in the long-term. The ongoing climate change appeared to work in opposite ways in this case because declining depth of snow cover will make the situation worse but increased precipitation will improve. We suggest that the main reasons for long-term decline of body condition of parent owls are interactive or additive effects of reduced food resources and increased overall predation risk due to habitat degradation (loss and fragmentation of mature and old-growth forests due to clear-felling) subsequently leading to decline of Tengmalm’s owl study population. Although changes in physiology  as a trait level responses are relatively easy to measure in relation to changing climate worldwide4, it is surprising that they are less studied than phenological traits5. It was reported earlier that warmer temperatures have been connected with reduced body condition in both juvenile and adult birds7. Responses in body condition to global warming are usual, but their direct influences on reproduction and then population growth seem to have only minor impacts regarding the total temperature on population dynamics5, which suggests that other unknown factors are probably involved. There also are evidences that weather conditions can directly influence densities of animal populations by alteration of reproductive success and/or survival, but the mechanisms of changes can also be indirect via modifying habitat and/or food abundance8. Recently, global changes in climatic conditions have been causing decreases in population densities of many different species, by worsening their reproductive success due, for instance, to phenological mismatches thus causing declines in population abundances which are in some causes leading up to local extinctions9.Physical condition of individuals is important for the ability to resist various parasites and diseases as well as in escaping predators thus contributing to reproductive success, over-winter survival and changes in wildlife population densities8. Outside the breeding season, organisms cope with energetic challenges such as, for instance, disease, dispersion, challenging weather, and reduced food availability that can reduce energetic reserves available for future breeding, and these energetically demanding activities occurring during one life history stage can carry over to impact performance in a later stages15. Body condition of bird of prey adults and their offspring ultimately depends on food abundance and maintaining body condition may be related to age or the ability to defend good-quality territories3. Usually, yearling birds are in poorer condition during breeding season than older, more experienced individuals17. Females in good condition laid early and larger clutches and their condition decreased as the season advanced21. On the other hand, individuals in poor condition may not breed at all in a given year under conditions of food scarcity22. Body condition has also been used as a measure of health for a number of bird species25. Body condition of nestlings in asynchronously hatching bird species has been shown to be related to the hatching order within the nest and to the number of hatched individuals/brood size27.Very little is known about the effects of long-term changes in weather conditions (climate-induced changes) on body condition of individuals on a population level from the long-term point of view, although food abundance in particular and also weather conditions are considered as key factors influencing the body condition, reproductive success, survival and population densities of birds and other animalsAegolius funereus) are small predatory birds with almost exclusively nocturnal activity. They breed in cavities but readily accept also artificial nest boxes. The boreal zone coniferous forests and also alpine forests further south in the Holarctic region are their main habitats29. Tengmalm’s owls feed mainly on voles of the genera Myodes and Microtus (main prey) and during vole scarcity on shrews and small forest bird species 33. In this species, breeding duties are clearly defined. Males are food providers to females and their young during the whole nesting period and also during the post-fledging dependence period which may last 5 to 9 weeks36. The mean lifespan of Tengmalm’s owl males is 3.5 years on average and mean breading lifespan is about 1.5 years37. Female incubates the eggs, take care of the nestlings, and remains in the nest until the offspring are about 3 weeks old38. The eggs hatch in ca 2-day intervals 30. For this reason, first-hatched young are on average 1 week older than last-hatched young27, which results in a marked size hierarchy among siblings. The duration of nestling period in individual young may take 27–38 days (from hatching to fledging), they leave the nest in the order as they hatched at intervals of about 1 day and the duration of fledging period increases with the number of siblings38.Tengmalm’s owls  body condition of offspring and parent owls has decreasing long-term trends, too. Because clutches and broods are larger in good vole years than in poor ones32, (ii) offspring and parent owls would also be in better body condition in years with abundant main foods (voles) than in those of scarcity of main foods. Further, because older individuals surviving first year of their lives will have more experience and will be better hunters32, we predicted that (iii) body condition of parent owls increases with their age. Finally, because early breeders are usually the fittest and most productive individuals in the population42, we predicted that (iv) body condition of offspring decreases with delayed laying date, but (v) increases with body condition of parent owls in the extension of the third prediction44.The nation-wide population of Tengmalm’s owls in Finland was declining by 2% per year from the early 1980s to late 2010s resulting in overall decrease of population by about 70% up to 201945). Tengmalm’s owls breed in the natural cavities made by black woodpeckers (Dryocopus martius) in Europe but readily accept nest boxes. Nowadays, there are ca 450 nest boxes, which we inspected annually during 1973–2018, in the study area covering ca 1100 square km45.We conducted the study in the Kauhava region of west-central Finland . This lowland study area is only 30–120 m above sea level and around 61% of the study area is forested. Nearly all the forest areas are managed: first harvested by thinning when trees are 30–40 years old, and thereafter clear-cutting at intervals of 60–80 years. Nowadays old-growth forests comprise around 1% of the area. Clear-cut and sapling areas accounted for ca 7% of the study area, and agricultural land (mainly crop fields and pasture) covered 25%, peatland bogs 2%, other  3%, and water  2% of the area (for more details see40). Long-term trends for weather variables and their individual periods used in particular analyses are presented in the Supporting Information file , total snow cover (cm), and daily precipitation (mm) during 1973–2018 (for details see30. We inspected the nests once per week (which is sufficiently often) until we found out the final number of eggs and hatchlings and to determine the hatching date (± 1 day). The age of the nestlings and their hatching order was based on the recorded date of hatching. All nestlings were ringed, and from 1985 onwards weighed and their wing length was measured at approximately the age of 25 days of the oldest owlet of the brood. We trapped a vast majority of parent females and males breeding in the study area during the middle of the nestling period. Parents were ringed, aged by checking the moult of primary and secondary feathers32, and we measured their wing length and body mass . Tengmalm’s owls can be reliably aged into three categories: 1-year, 2-year and older (3+) owls32.We visited wooden nest boxes in the study area in late March to late April and again in May to early June to find nests. The date of the laying of the first egg was obtained mainly by back-dating from hatching dates using 29 days as the incubation period for the first-laid egg46 was calculated to quantify the body mass relative to the body size of owls. The regression slopes were 0.60 for offspring, 0.84 for males and 0.77 for females, whereas the average (± SD) wing lengths were 100.1 ± 19.83, 171.7 ± 3.69 and 178.9 ± 4.16 mm, respectively. Thus, we calculated the scaled mass index Ĥi  as follows: Ĥi = Hi (L0/Li)bSMA where Hi and Li are the body mass and the linear body measurement of individual i, respectively; bSMA is the scaling exponent estimated by the standardised major axis (SMA) regression of H on L; L0 is an arbitrary value of L ; and Ĥi is the predicted body mass for individual i when the linear body measure is standardized to L0 according to Peig and Green46.The “scaled mass index” following the method by Peig and GreenMyodes glareolus, field voles Microtus agrestis and sibling voles Microtus rossiaemeridioinalis) in the study area by snap-trapping each year in early May and in mid-September. Sampling was carried out in the four main habitat types . Fifty-to-sixty baited Finnish metal mouse snap traps were set at 10 m intervals in vole runways on each sample plot and were checked daily for 3 consecutive days. The area of a sample plot was 0.5–0.6 ha, and the pooled trapping effort was approx. 600 trap-nights each year and season starting in 1973. The number of voles captured was standardized to the number of animals caught per 100 trap-nights (see47 for more details on trapping methods and vole cycles in the study area). As found earlier48, densities of bank and Microtus voles fluctuate in synchrony in the study area and the regional synchrony of vole population cycles extends up to 80 km, i.e., to the whole study area.We estimated abundances of main prey of Tengmalm’s owls . The methods were carried out in accordance with the relevant guidelines and regulations of the Finish Museum of National History.40 when we analysed questions regarding the breeding performance of Tengmalm’s owls such as breeding densities, timing of breeding, clutch size and number of fledglings per breeding attempt. In the current study, we added body condition indices of parent owls and offspring in the dataset, and thus analysed the crucial second part of the whole long-term study.A majority of the dataset used in this study was already published elsewhere40. The two variables and their timespan (period) chosen fitted best our a priory hypotheses, were in line of the previous study, and other different periods were not tested compared to the previous analyses40. We first explored the correlations between the Tengmalm’s owl study population variables involved. Significant correlation was found between the number of eggs (E), hatchlings (H) and fledglings (F)—. We subsequently made a judgment of the extent of collinearity by checking related statistics, such as Tolerance value or Variance Inflation factor (VIF), Eigenvalue, and Condition Number following the approach of Schreiber-Gregory and Jackson49 and using TOL, VIF and COLLIN options of the MODEL statement in the SAS REG procedure. The lowest Tolerance value was 0.20949 and the highest VIF value was 4.77340. The relationship of the Eigenvalues (range 0.11965 to 1.59292) to the Condition Index values (range 4.54237 to 1.24493). So, there was no threat of multicollinearity indicated through these diagnostic results. Correlations are generally considered “strong” above 0.849 or at least 0.550. To avoid any possible interdependency, we still considered the number of eggs, hatchlings and fledgelings redundant so that only one of these three variables entered a statistical model.We analysed the data with the aid of SAS System version 9.4 (SAS Institute Inc.) in three steps. The analysis checking for possible multicollinearity was done separately for the Tengmalm’s owl study population data , weather , and prey abundance data (abundance of main prey in current spring and previous autumn). The two weather variables used in the three models  were chosen based on the analyses and results of the above-mentioned study partly dealing with the same data setWe applied the same procedure for checking possible multicollinearity for mean of daily precipitation (mm) during May–June, mean of total snow cover (cm) during January–March, and abundance of main prey (voles) in current spring and previous autumn. There was only a high correlation between the abundance of main prey (voles) in the current spring or previous autumn . Therefore, only one of the two variables was used in further analyses. The lowest Tolerance value was 0.73587, and the highest VIF value was 1.35893. The relationship of the Eigenvalues (range 0.00357 to 0.22216) to the Condition Index values (range 32.10814 to 4.07254). Thus, neither in the weather and prey abundance data there was no indication of possible multicollinearity.51. We used non-temporal analytic framework for our statistical analysis instead of autocorrelation approach because mean breading lifespan of Tengmalm’s owls is about 1.5 years37, which does not allow meaningful autocorrelation analysis.Second, using three different models Table , a–c ana52 and prepared a priori multiple hypotheses based on the remaining biologically relevant variables after testing for collinearity 56. Multiple information criteria are useful because each one was developed to optimize something different than the others. AIC is an example of efficient information criteria, while BIC is an example of consistent information criteria57. We found justification for such a procedure in a study of Posada and Buckley58. They showed that AIC and BIC are able to simultaneously compare multiple nested or non-nested models and assess model selection uncertainty52.Third, we applied model selection based on the information-theoretic paradigm using Akaike’s Information Criterion—IT-AICi) between the Fit statistic values  were sorted according to AIC values. Akaike weight wi can be interpreted as the probability that Mi is the best model , given the data and the set of candidate models59. For five models with the lowest AIC values, we therefore calculated Δ AIC, Akaike weights wi, and for estimating the strength of evidence in favour of one model over the other we divided their Akaike weights wmin/wj (AIC Odds)59. As recommended by various authors61, using the same formulas just replacing AIC by BIC values, we obtained analogically Δ BIC, BIC weights wi, and BIC Odds. The advantage of this is that in comparison with AIC, BIC severely penalizes models with more parameters. Thus, the BIC weights wi are appreciably different than for AIC weights wi60.The differences  and a relative information loss [exp((AIC_null − AICi_best)/2)], an approach adapted from Burnham and AndersonFinally, having the best composition of all models according to IT-AIC, these were calculated using GLMM, coefficient estimates, standard errors, and 95% confidence intervals were calculated for each model, and the results generated into the graphs.In total, we recorded 1761 nests during 1973–2018 and handled  1171 male parents during 1979–2018, 1468 female parents during 1977–2018, and 3971 offspring in 1044 broods during 1985–2018.wi and AIC Odds revealed comparable if not even identical results with Δ BIC, BIC weights wi and BIC Odds. This strengthened the credibility of the results. Comparison of the best model to the null model for all dependent variables is in Table Table The composition of factors of the model with the lowest AIC and BIC values for the BCI of offspring had essential support with the probability of 87% that it is the best model Table . The modThe best model explaining BCI of offspring Table  includedThe composition of factors of the model with the lowest AIC and BIC values for the BCI of male parents had essential support with the probability of 85% that it is the best model Table . The modThe best model explaining BCI of male parents Table  includedThe composition of factors of the model with the lowest AIC and BIC values for the BCI of female parents had essential support with the probability of 78% that it is the best model Table . The modThe best model explaining BCI of female parents Table  includedThe main findings of this study were that body condition of both male and female parents of Tengmalm’s owls showed declining long-term trend, whereas the body condition of their offspring slightly increased from 1980 to 2010s. In addition, depth of snow cover emerged as the most important weather variable modifying the body condition of parent males and females, as well as the amount of precipitation appeared as the most important factor affecting the body condition of offspring in the long-term.40. The main reason for marked decline in offspring production was brood reduction due to lack of food inducing starvation of owlets during the nestling period. Valkama et al.27 showed that the proportion of hatchlings producing fledglings decreased with the degree of asynchrony within broods during the decrease phase of the vole cycle, which showed that nestling mortality was most frequent among asynchronously-hatched broods when food became scarce. Therefore, when owls raised overall smaller broods these offspring could consequently be in slightly better condition. This result is consistent with Lack’s62 brood reduction hypothesis stating that if food becomes scarce during the nestling period the youngest nestlings would die first without endangering the survival of the whole brood. This further corresponds well with the result that body condition of offspring decreased with increasing number of hatchlings, as it was also found, for instance, in burrowing owls (Athene cunicularia)26.Contrary to our expectations, body condition of offspring was very slightly increasing  throughout the study period. This result might be explained by the fact that the fledgling production per breeding attempt by the same parent owls decreased during the same time period40. It was earlier found that increased summer temperatures improved vole densities and maintained 3-year high-amplitude cycles of vole populations in South and Central Finland63. Warm and humid spring and summer seasons have positive effects on vole densities via improved food supply of herbivorous voles, and thus increase both body condition and offspring production of Tengmalm’s owls40.The finding that body condition of offspring increased with increasing amount of precipitation during the current spring (May to June) is consistent with our previous result that the number of fledglings produced per breeding attempt also augmented with the amount of precipitation during the same time periodFalco tinnunculus) from hatching onwards. Offspring of both species were in better body condition in supplementary fed nests than in non-supplemented control nests64. These results are consistent with results on recruitment rate of Tengmalm’s owl offspring to the future breeding population, which is two-to-three times higher for fledglings hatched in the increasing phase of the vole cycle with improving conditions of main foods than in the decrease and low phases of the vole cycle with deteriorating or poor abundances of main foods65. Similarly, in passerine birds better food availability during the nestling period improved growth, development, body condition and survival of nestlings, and can also positively impact post-fledging survival and recruitment probability72, but contradictory findings were also reported75. In a similar way, better body condition, post-fledging survival and recruitment probability thanks to higher food availability was also recorded in several bird of prey species80, but not in Ural owls  subsisting mainly on voles1. Thus body condition at fledging obviously have carryover effects in post-fledging survival which in turn is crucial for reproductive success with ultimate consequences for individual fitness and population dynamics82.As expected, the body condition of offspring amplified with augmenting abundance of main foods in the current spring. This result reveals the importance of limitation in main food resources during the nestling period for body condition of offspring, and thus for their future survival. This interpretation is also supported by the results from supplementary feeding experiments in both Tengmalm’s owls and Eurasian kestrels  hoarded more food items in their winter food stores and during low vole years stored less birds  than the adults91. This suggests that less-experienced yearlings rely more on stored food than adults90, and thus supports the explanation that experienced individuals are better able to capture some prey on the daily basis as mentioned above. Alternatively, this result might arise from the fact that yearling owls, particularly yearling males, usually breed only in good vole years32, thus being in better body condition compared to older individuals breeding also during declining and/or low phase of the vole cycle and being most probably in worse body condition at that time. Further, we should keep in mind that the parent owls were trapped and weighed during the mid-nestling period in spring. It is simply possible that we could find the opposite (older owls to be heavier than yearlings) in the middle of winter (when it is better to have more fat reserves) as found, for instance, in snowy owls (Bubo scandiacus)3.Contrary to our expectations, the body condition of parent owls was slightly decreasing with their age. We can speculate the older and more experienced individuals do not need to make body reserves as large as yearlings because they are better able to capture some prey on the daily basis. This can be supported by the fact that adults had greater hunting success than juveniles in nine raptor species40. We suggest that deep snow cover in the course of winter offers effective insulation for over-wintering voles that can even reproduce below the deep snow cover92, where the ambient temperature is relatively constantly close to 0 °C. Deep snow cover may thus have a positive effect on overwinter survival of voles95, and may result in higher vole densities in early spring, which in turn may induce larger clutch sizes and better body condition of parent owls.We further found that body condition of parent owls increased with increasing height of snow cover during the preceding winter (January to March) similarly as we previously found for clutch size of Tengmalm’s owls40. The ongoing climate change appeared to work in opposite ways in this case because declining depth of snow cover will make the situation worse , whereas increased precipitation will improve the situation . We suggest that the main reasons for long-term decline of body condition of parent owls are probably interactive or additive effects of reduced food resources and increased predation risk due to habitat degradation: loss and fragmentation of mature and old-growth forests due to clear-felling. Degradation of forest habitat subsequently leads to decline of local Tengmalm’s owl population40 and nation-wide population in Finland96. It seems that Tengmalm’s owls are not able to cope with multiple stressors induced by forest habitat degradation which have negative carry-over effects in the long-term regarding particularly parents’ body condition and breeding success, and most probably also over-winter survival. In addition, the situation is also quite similar and serious for many other forest-dwelling specialists inhabiting boreal forests104, and it will only deteriorate over time unless major measures to reverse the degradation of forest habitat due to clear-felling are taken and actually implemented as soon as possible.We conclude that the decreasing trend of body condition of parent owl males and females is important factor probably inducing reduced adult survival, as well as reduced reproduction success thus contributing to the long-term decline of the Tengmalm’s owl study population. The very slightly increasing trend of body condition of offspring is obviously not able to compensate the overall decline of Tengmalm’s owl population, because the number of fledglings in turn simultaneously decreased considerably in the long-termSupplementary Information.


Background: Lcz696  shows an inhibitory effect on fibrosis after myocardial infarction (MI). However, the underlying signaling mechanisms are poorly understood. The Wnt/β-catenin signaling pathway is activated after MI and participates in the process of myocardial fibrosis. Here, we aimed to assess the efficacy of ARNI for alleviating myocardial fibrosis after MI and hypothesized that ARNI alleviates myocardial fibrosis by inhibiting the Wnt/β-catenin signaling pathway and overexpressing sFRP-1, an inhibitor of the Wnt/β-catenin signaling pathway.Methods: Mice randomized at 1 week post-MI were administered lcz696 , valsartan , or corn oil (n = 13) orally for 4 weeks, while the sham-operated group received vehicle . Cardiac function and extent of myocardial fibrosis were measured. Western blotting and quantitative real-time polymerase chain reaction were used to detect the expression of Wnt/β-catenin pathway-related proteins. Furthermore, primary myocardial fibroblasts were stimulated with angiotensin II (Ang II) and cultured with lcz696 and the sFRP-1 inhibitor way316606 to detect the expression of Wnt/β-catenin pathway proteins.Results: Both lcz696 and valsartan alleviated myocardial fibrosis and improved cardiac function, but lcz696 had superior efficiency compared to valsartan. Furthermore, β-catenin expression was inhibited and sFRP-1 was overexpressed after drug treatment, which could be significantly improved by lcz696 in mice. In addition, lcz696 inhibited β-catenin expression in AngII-stimulated myocardial fibroblasts, and β-catenin expression increased after the inhibition of sFRP-1.Conclusion: ARNI alleviated cardiac fibrosis and cardiac remodeling by inhibiting the Wnt/β-catenin signaling pathway. In addition, ARNI can lead to overexpression of sFRP-1, which is an inhibitor of the Wnt/β-catenin signaling pathway. These results indicate a new therapeutic target of ARNI to improve myocardial fibrosis and prevent myocardial remodeling. Heart failure following acute myocardial infarction (MI) remains a major public health concern worldwide, exerting a substantial economic burden . CardiacRegarding the underlying molecular mechanisms, ample studies have recently shown an emerging role of the Wnt/β-catenin signaling pathway in the process of cardiac remodeling; inhibition of this pathway has been shown to be cardioprotective, to avert cardiac inflammation and fibrosis, to reduce infarct size and scaring, and to stimulate functional recovery of the heart .Wnt signaling pathways are considered essential in heart development and are found to be active in the post-MI heart . Wnt/β-cThe secreted frizzled-related protein 1 (sFRP-1), an antagonist of the Wnt/β-catenin pathway , has beein vitro and in vivo. Particularly, we comprehensively investigated the relationship among ARNI, the Wnt//β-catenin pathway, and sFRP-1.Mechanistically, the beneficial impact of ARNI may partially stem from the inhibition of Wnt/β-catenin signaling. Based on previous studies, we hypothesized that lcz696 could be implicated in post-MI myocardial fibrosis by regulating the Wnt/β-catenin signaling pathway. However, the correlation between ARNI and sFRP-1 has not been studied thus far. Therefore, herein, we evaluated the effects and mechanisms of ARNI in the context of MI, both All mouse studies were approved by the Animal Ethics Committee of Shandong University; the care and use of animals followed the guidelines on animal ethics. Eight-week-old C57BL/6J male mice were purchased from SBF Biotechnology Co. Ltd. . The mice were housed at a constant temperature (24°C) and provided a normal diet with free access to water. C57BL/6J male mice underwent either a permanent coronary artery ligation to induce MI or sham surgery. Briefly, the left coronary artery (LCA) was positioned using a 6–0 silk suture, stitched, and ligated approximately 3 mm from its starting point . When thMice were anesthetized via inhalation of isoflurane gas; induction was performed at 2.5% in a chamber, and maintenance was carried out at 1.5% isoflurane via a nose adaptor. The cardiac function was then evaluated using the Vevo770 imaging system . The left ventricular ejection fraction (LVEF), fractional shortening (FS), early (E) peak, late (A) peak, left ventricular end-diastolic dimension (LVEDd), and left ventricular mass (LV mass) were measured. The ratios of early-to-late mitral flow velocity (E/A) and diastolic velocity ratio (E’/A’) were also calculated. Additionally, the internal diameter of the LV and the thickness of the septum and posterior wall at end-systole and end-diastole were measured from the long-axis view at the level of chordae tendineae. LVEF and FS were then calculated according to these parameters. Echocardiographic imaging and measurements were performed in a blinded manner. All measurements were averaged based on three consecutive cardiac cycles.The serum levels of NT-proBNP were measured using a commercial enzyme-linked immunosorbent assay (ELISA)—Mouse NT-proBNP ELISA Kit , according to the manufacturer’s instructions. Briefly, this assay employs a quantitative sandwich enzyme technique. A microplate is pre-coated with an antibody specific for NT-proBNP; the analyte is captured, and then sandwiched with a biotin-conjugated antibody. Thereafter, avidin-conjugated horseradish peroxidase is added, followed by a specific substrate. Finally, color development is stopped, and the intensity of the color is measured. In our assay system, the intensity of the developed color was positively correlated with the concentration of mouse NT-proBNP in the sample, and the absorbance  was measured with using an absorbance microplate reader  at 450 nm wave length.The systolic and diastolic blood pressure of awake mice was measured using the tail-sleeve method before and after surgery and before death. The heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were measured using a noninvasive tail-cuff system .Animals were humanely euthanized, and their hearts were quickly removed, rinsed with normal saline, and dried. Heart tissues were frozen in a −20°C refrigerator for 30 min until they hardened. Then, from the apex of the heart to the bottom, 1–2 mm thick sections were obtained, along the direction of the atrioventricular groove. A total of five slices were cut. The slices were quickly placed into a TTC phosphoric acid buffer solution  at 37°C, and were subsequently bathed in water for 15 min. The infarcted area after staining appeared white, while the normal areas appeared red.Mice were anesthetized and euthanized, and their hearts were dissected; the dissected tissues were immediately fixed with 4% formalin. Tissues were then embedded within paraffin and sectioned (5 µm) for histology staining including hematoxylin and eosin (H&E) and immunohistochemistry. Of note, the cardiac tissues were removed from 3 to 4 mm below the ligation site to ensure that the pathological sections of cardiac tissues were at the same level. For the detection of interstitial collagen deposition, heart sections were stained with PicroSirius Red and Masson’s trichrome according to the manufacturer’s instructions. PicroSirius Red staining images were captured at 10× on a BX51 epifluorescence microscope  using circularly polarized light to generate a birefringent (red/thick and green/thin) signal from collagen fibers. To evaluate fibrosis, the areas of interest  were defined using ImageJ. The fraction of the fibrosis area was quantified using ImageProPlus6.0 .For immunohistochemistry, the slides were incubated overnight at 4°C with specific primary antibodies against SFRP-1 , β-catenin , GSK-3β , collagen I , and collagen III . The next day, the samples were incubated with secondary antibodies and diaminobenzidine staining  was performed following the manufacturer’s protocols. Data were analyzed using ImageProPlus6.0 .in situ cell death detection kit  following the manufacturer’s instructions. Heart sections were deparaffinized, hydrated, kept in permeabilization solution (0.1% Triton X-100) for 5 min, and incubated in TUNEL reaction mixture for 60 min at 37°C; the sections were then sealed in Prolong Gold Anti-Fade Reagent with 4-6-diamidino-2-phenylindole . Images were acquired via a fluorescence microscopy (Nikon) and quantified using ImageProPlus6.0 software .Apoptotic cells in myocardium were detected using a Heart sections were incubated with specific primary antibodies against α-SMA  at the appropriate concentrations at 4°C overnight. The next day, horseradish peroxidase-conjugated secondary antibodies were added for 1 h at 37°C. Nuclei were visualized using DAPI staining. Fluorescent images were acquired via a fluorescence microscopy (Nikon) and quantified using Image Pro Plus 6.0 software .2 humidified incubator at 37°C for 36 h. Before lcz696 or VAL stimulation, the cells were starved in serum-reducing medium (2% FBS) for 12 h. Subsequently, we added different lcz696  in varied concentrations   100 nmol/L requires 48 h   and incures 48 h . Therefo−ΔΔCt comparative quantification method  following the manufacturer’s protocol, and the conversion of mRNA into cDNA was performed using the Primescript™ RT reagent kit with gDNA Eraser . qRT-PCR was performed using SYBR Premix Ex TaqII  in the iQ5 Multicolor Real-Time PCR Detection System  using gene specific primers . Relativn method . All expTotal protein from mouse myocardium tissues and primary cardiac fibroblasts was extracted using a protein extraction kit and protein concentration was measured using BCA Protein Assay kit  according to the manufacturer’s protocol. The proteins were separated by 10% gradient SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane using the wet transfer method. The membranes were blocked with 5% defatted milk for 1 h at room temperature and then incubated overnight at 4°C with specific primary antibodies against sFRP-1 , β-catenin , active β-catenin , GSK-3β ,p-GSK-3β , α-tubulin , collagen III  and α-SMA . All primary antibodies are diluted in proportion with antibody diluent (Boster). The membranes were washed and incubated with the anti-mouse/rabbit secondary antibodies labeled with horseradish peroxidase  for 1 h at room temperature. The blots were subsequently detected using a chemiluminescence kit  as per the manufacturer’s instructions. All experiments were repeated at least three times.t-tests (two-group comparisons), or the one-way ANOVA followed by Dunnett’s or Tukey’s post hoc tests (comparisons between multiple groups). p < 0.05 was considered statistically significant.All data are expressed as mean ± SEM derived from at least three independent experiments. Statistical analysis was performed using GraphPad Prism 8 . The Shapiro-Wilk test was applied for normality assumption. Thereafter, normally distributed data were analyzed using the unpaired two-tailed Student’s C57BL/6J male mice underwent permanent LCA ligation to induce MI. Importantly, TTC staining was performed 24–48 h after MI and showed that the mouse model of MI was successfully established with the infarcted areas appearing in white; of note, no infarcted areas were detected in sham-operated animals . AdditioThe serum NT-proBNP levels were significantly increased 5 weeks after MI in the MI group; of note, there was a significant difference upon treatment with ARNI and VAL, and the effect of ARNI was superior to that of VAL . MeanwhiMasson’s trichrome and PicroSirius red staining of heart sections demonstrated that collagen deposition in the hearts of MI animals was worse than that observed in the sham group, as expected. Additionally, ARNI treatment prevented collagen deposition to a greater extent than VAL treatment . We alsop-GSK-3β were the opposite . Interestingly, after 4 weeks of treatment with ARNI and VAL, the expression of β-catenin was inhibited; of note, the expression of β-catenin and active β-catenin in the ARNI group was significantly lower than that in the VAL and MI groups . In contopposite . Similaropposite . Althougopposite , there wopposite .Next, we investigated the expression of sFRP-1, which is found upstream of the Wnt signaling pathway. The western blot results showed that the expression of sFRP-1 was increased in the MI group compared with that in the sham group. Remarkably, after drug treatment, the expression of sFRP-1 was increased more obvious; of note, the increase was observed to be higher in the ARNI group than in the VAL group . Importa+) was higher than that in lcz696-treated cells and lower than that in AngII-treated cells, suggesting that the influence of lcz696 on β-catenin is related to the expression of sFRP-1 (For further mechanistic insights, we obtained primary mouse fibroblasts according to the methods mentioned above and studied the effects of lcz696 and VAL on the Wnt/β-catenin signaling pathway as well as on the expression of sFRP-1. The western blot results indicated that β-catenin expression was significantly inhibited at 30 mmol/L lcz696 . The qRTf sFRP-1 . The resf sFRP-1 . Additiof sFRP-1 . In addif sFRP-1 , suggestIn this study, we show that the sFRP-1/Wnt/β-catenin axis plays a role in the prevention of myocardial fibrosis and improvement of myocardial remodeling in the context of ARNI treatment.Studies have shown that ARNI has an inhibitory effect on fibrosis after MI, but the exact underlying mechanism is not entirely clear. Cardiac fibrosis is a common process in remodeling hearts after MI and is mediated by myofibroblast invasion and collagen deposition . In the ARNI was observed to show significant improvement over standard of care therapy in patients with class II, III, or IV heart failure and reduce the ejection fraction in the PARADIGM-HF trial . In the The Wnt signal transduction pathway is involved in myocardial repair after MI . Schuma. Studiesp-GSK-3β was different from the results of previous studies (Under general physiological conditions, the Wnt/β-catenin signaling pathway is inactive, and β-catenin is phosphorylated by the GSK-3β degradation complex following phosphorylation, β-catenin is identified and degraded by the ubiquitin-mediated proteasomal degradation pathway, maintaining low levels of expression in the cytoplasm. The canonical Wnt signaling pathway is activated after MI , protein studies , which m studies  or likelthis enzyme is responsible for the phosphorylation of β-catenin . Inhibitin vivo. We speculated that the inhibition of the Wnt/β-catenin pathway by ARNI might be achieved through sFRP-1. To further verify our hypothesis, we stimulated myocardial fibroblasts with AngII in vitro, after pre-incubation with lcz696 and Val. Interestingly, the expression of β-catenin was the lowest when 30 μmol/L of ARNI was administered. Additionally, the inhibition of sFRP-1 led to an increased expression of β-catenin. These observations provide novel insights into the mechanism by which inhibition of the Wnt/β-catenin signaling pathway by ARNI might be achieved by influencing the expression of sFRP-1.Although studies have shown that inhibiting the Wnt pathway leads to cardioprotective effects in the context of MI in mice, blocking the Wnt signaling pathway has been shown to lead to an increased heart rupture rate . BarandoAlthough our study provides new insights into the mechanism of action of ARNI in myocardial fibrosis, the underlying molecular mechanisms are not well understood. Further investigation is required to explore the specific mechanism of the regulation of the Wnt/β-catenin signaling pathway by ARNI.This study confirmed that the Wnt/β-catenin signaling pathway is activated in the context of MI. Importantly, we show that ARNI improves myocardial fibrosis, prevents myocardial remodeling, and inhibits the Wnt/β-catenin signaling pathway via the upregulation of sFRP-1 in the context of MI.


The purpose of this study was to determine the effect and mechanism of alkaline fertilizer, bio-control fungi, and their synergistic application on control of Fusarium Tr4 incidence. Synchronized use of the alkaline fertilizer and biocontrol fungi eliminates rhizome browning and reduces the incidence rate of banana Fusarium wilt. The incidence of yellow leaves  and disease index in +Foc Tr4 CF treatment were the same (65%), while incidence of yellow leaves and disease index in +Foc Tr4 AFBCF were 31% and 33%, respectively. Under the stress of Foc Tr4 infection, the synergistic utilization of the alkaline fertilizer and biocontrol fungi would raise the activities of peroxidase, catalase and superoxide dismutase in banana roots. The root activity of banana was also increased. As a result, the banana height and stem diameter increments, shoot and root dry weight, accumulation of N, P and K in banana plants had been increased. The efficacy of the synergistic application of alkaline fertilizer and biocontrol fungi was not only reducing Foc Tr4 pathogen colonization and distribution in banana plants, but also preventing tylosis formation in vascular vessel effectively. Therefore, the normal transport of water and nutrients between underground and aboveground is ensured.  Musa spp. AAA) is of great importance to the world food security as a fruit and food crop [Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc Tr4) [According to FAO statistics, banana (ood crop . Howeverood crop . The banFoc Tr4) . It is hFoc Tr4) . Thus, oThe most widely used methods to control banana Fusarium wilt are chemical and agricultural measures as well as disease resistant variety release. The chemical control includes quaternary ammonium compounds, soil disinfectants, propiconazole and the other chemical agents which are used to kill pathogens in the soil directly ,7,8. Thenon-pathogenic Fusarium oxysporum, Trichoderma and Paecilomyces lilacinus could significantly reduce the occurrence of banana Fusarium wilt. The results imply that the combination of the three fungi has a synergistic effect [Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc Tr4) in banana plants were studied by green fluorescent protein (GFP)-tagged Foc Tr4. The findings would be expected to provide a theoretical foundation for the coordinated control of banana wilt with alkaline fertilizer and BCF.Soil acidification which is caused by long-term excessive and irrational use of chemical fertilizer, especially nitrogen fertilizer in banana plantation results in the proliferation and spread of banana wilt pathogens ,19,20. Ic effect . Howeverc effect ,22. It cMusa spp. AAA) was used in this study. Banana seedlings with 5 green leaves and average height of 5–6 cm was used to carry out sterilized sand culture experiment. Average plant height and stem diameter of plantlets were surveyed before transplanting. Cavendish banana cultivar Brazilian  and provided by the South China Agricultural University fungal laboratory. The biocontrol fungi (BCF) applied in the trail was composed of 2O content accounted for 60%) and sodium dihydrogen phosphate (P2O5 content accounted for 45%) into urea-formaldehyde solution during the urea-formaldehyde formation. N:P2O5:K2O ratio of the liquid fertilizer was 2:0.5:1. The fertilizer was liquid fertilizer, which included conventional fertilizer and alkaline fertilizer. The pH of the liquid fertilizers was 5.5–6.0 and 7.5–8.0 for conventional fertilizer and alkaline fertilizer, respectively. The fertilizer was prepared by adding urea (N content accounted for 46%), potassium chloride  and non-inoculation pathogen (−Foc Tr4). The BCF included added and without added BCF. The fertilizer included conventional fertilizer and alkaline fertilizer. The eight treatments were defined as −Tr4 CF, −Tr4 AF, −Tr4 CFBCF, −Tr4 AFBCF, +Tr4 CF, +Tr4 AF, +Tr4 CFBCF, +Tr4 AFBCF, respectively. Thirty replicates were designed for each treatment, and one pot was one replicate. All treatments received the same amount of N, P and K. The N applied to each treatment was 0.2 g per kilogram sand. The fertilizer was dissolved in water and divided into 10 times to be applied once a week. The pH of the fertilizer solution was adjusted to 5.5–6.0 and 7.5–8.0 for conventional fertilizer and alkaline fertilizer before application, respectively. non-pathogenic Fusarium oxysporum, Trichoderma (Trichoderma harzianum QL18-8) and Paecilomyces lilacinus were inoculated in Potato Dextrose Agar (PDA) medium, respectively. The fungus was activated under 26 °C condition for 7 days. Twenty fungi plugs of 8 mm in diameter were placed in 500 mL Potato Dextrose Water (PD water), respectively. After shaking 7 days at 180 rpm under 26 ± 0.5 °C, the fungus suspension was then filtered through eight layers of gauze to separate the hyphae. Then, the filtrate of non-pathogenic Fusarium oxysporum, Trichoderma and Paecilomyces lilacinus were diluted with sterile deionized water until the number of spores of each fungus was 3 × 107 conidia/mL, respectively. Then the three diluted filtrates were mixed in a volume ratio of 1:1:1 to form the BCF.The 5 conidia/mL, which was the Foc Tr4 pathogen for inoculation.Pathogen of Foc Tr4 was inoculated in PDA solid medium and activated under 26 °C for 7 days. Twenty fungi plugs of 8 mm in diameter were placed in 500 mL PD water. After shaking 7 days at 180 rpm under 26 ± 0.5 °C, the fungus suspension was then filtered through eight layers of gauze to separate the hyphae. Then, the filtrate was diluted with sterile deionized water until the number of spores was 105 conidia/mL was inoculated as above. The mock-inoculated control plants were also treated with 30 mL sterilized water to each pot.Healthy banana plantlets with similar height, diameter of pseudostem and same number of green leaves were selected. Roots of banana plantlets were immersed in 1000 times diluted chlorothalonil solution for a few seconds before transplanting. The banana plantlets were then inoculated with the BCF four weeks after transplantation by use of injured root inoculation method as follows. Four 5 cm deep holes were made in four directions of banana plantlets by use of sterilized glass rod. Thirty milliliter the BCF solution was poured into the four holes evenly, and the holes were covered with sand. The mock-inoculated control plants were treated with 30 mL sterilized water to each pot. Ten days later 30 mL of GFP-tagged Foc Tr4 spore suspension at 10After inoculation of Foc Tr4, disease infection state of the banana was recorded by observation of leaf color change every 10 days and then the IYL and DI was calculated. Banana Fusarium wilt was divided into 6 levels . Level 0Three banana plants were randomly selected from each treatment 60 days after inoculation. Rhizome and pseudostem of the banana were cut into two parts longitudinally. Image of the rhizome and pseudostem section was taken by WinRhizer Sysytem. The browning area of rhizome was calculated by Image-Pro (MEDIA CYBERNETICS).Five plants were randomly selected from each treatment to measure banana height and diameter of pseudostem 60 days after inoculation, and increment of the height and pseudostem diameter accounted. Banana roots, pseudostem and leaves were collected after the banana plant was poured out and sand attached to the root washed. Fresh weight of the roots, stems and leaves was measured, respectively. Samples were then headed at 105 °C for 30 min and dried at 75 °C to constant weight to account dry matter. The plant samples were stored for NPK test.Three banana plants were randomly selected from each treatment 60 days after inoculation, and roots were collected as above. Root activity was measured by the triphenyl tetrazolium chloride method (TTC method) according to Liu et al. . The ant2O2)-sulfuric acid (H2SO4) [The above dried roots, stems and leaves powder sample (passed to 0.25 mm sieve) was digested by hydrogen peroxide (H (H2SO4) . N, P anThe root 1 cm from rhizome, middle section of rhizome and three sections of pseudostem  of the plants inoculated with Foc Tr4 were examined for infection process and colonization at early stage of banana wilt disease. For microscopic observation, banana tissues were washed in sterile distilled water, then cut into thin sections along the vertical and longitudinal axes by hand sectioning. Then, the tissue sections were placed on a microscope slide, submerged in a water droplet, and covered with a glass cover slip. Microscopic observation was carried out under a Double-Scanning Laser Confocal Microscope (NIKON A1) equipped with filter blocks with spectral properties matching the fluorescence of GFP by taken Honghong Dong et al. as reference . Three pThe SEM samples were prepared according to Nebesářová’s method . Root saResults statistics were completed with Design-Expert 13. The data in tables and figures are mean ± SE, where SE is the standard error. The difference among treatments was estimated by Univariate ANOVA and multiple comparison.Bananas inoculated with Foc Tr4 showed obvious symptoms of wilt disease two months later, while bananas without Foc Tr4 inoculation grew healthily. When banana plants suffered banana wilt, their old leaves started yellowing and wilting and the symptoms gradually spread to new leaves. For severe symptoms, new leaves turned yellow and died. The number of banana leaves of +Tr4 CF treatment was the least, and all the leaves except the top one were withered and yellow. The bananas of +Tr4 AFBCF treatment showed less Fusarium wilt symptoms. Only old leaves were yellowing in comparison with those of Foc Tr4 inoculation treatment .The incidence of yellow leaves (IYL) and disease index (DI) of banana were significantly decreased by the combination use of alkaline fertilizer and BCF. As shown in p = 0, 0.057, 0.041, 0.05, respectively). No matter how the banana was inoculated Foc Tr4 or not, the height and stem diameter increments, shoot and root dry weight were increased when the BCF applied with fertilizer together (p < 0.05). The corresponding values of AFBCF were 47.9%, 158.5%, 59.0% and 50.7%, respectively (p < 0.05). The results showed that the synergistic effect of the BCF and alkaline fertilizer on banana growth was more significant than that of single BCF or its combination with conventional fertilizer. In the four treatments of −Foc Tr4, the banana height and stem diameter increments, shoot and root dry weight of AFBCF treatment were increased by 30.4%, 61.2%, 64.2% and 93.1%, respectively, compared with those of CF  in −Foc Tr4, respectively. The results indicated that the synergistic effect of fertilizer and BCF on banana root activity was greater when there was no pathogen infection. In summary, the root activity of banana plant would be decreased when the banana was infected by the Foc Tr4 pathogen. The synergism of the fertilizer and BCF could increase the root activity, and the synergistic application of alkaline fertilizer and BCF was superior to that of conventional fertilizer.Banana root activity was affected by inoculation of the Foc Tr4 pathogen. As shown in p < 0.01), respectively. In other words, after banana was threatened by the Foc Tr4 pathogen, the antioxidant enzyme activity could be increased. The application of fertilizer and BCF together would result in increase in the POD and SOD activity of banana root, regardless of whether the pathogen was inoculated or not (p < 0.05). Thus, the antioxidant enzyme activity of banana root was increased after the plant was stressed by the Foc Tr4 pathogen. The synergistic effect of the fertilizer  and BCF on the enzymes was more significant. The POD, CAT and SOD activity increment of banana root was from 4.18% to 36.62% in +Foc Tr4 treatments. The POD, CAT and SOD activity of AFBCF treatment were the biggest one among the four treatments when the banana suffered with Foc Tr4 stress (4 stress . In concp = 0.005). The N absorption and accumulation by banana could be improved by combination use of fertilizer and the BCF in case with or without Foc Tr4 inoculation. Without Foc Tr4 inoculation, the N accumulation of the combination use of fertilizer and the BCF was increased by 39.0% compared to that of BCF and fertilizer alone. However, with the Foc Tr4 inoculation, the corresponding value was 32.7% (p < 0.05), respectively. However, with Foc Tr4 inoculation, the N accumulation of CFBCF and AFBCF was increased by 48.4% and 74.9% (p < 0.05), respectively. The synergistic application of alkaline fertilizer and BCF was greater due to their combination could resist the damage of Foc Tr4 to banana growth. More N absorbed and accumulated by banana plant would promote its health growth and to resist the disease in turn. The results showed that the synergistic effect of alkaline fertilizer and BCF on eliminating banana Foc Tr4 infection was that the combination of alkaline fertilizer and BCF improved plant nitrogen nutrition and promoted banana health growth to resist the disease.Nitrogen (N), phosphorus (P) and potassium (K) nutrition state reflects the effect of alkaline fertilizer and BCF on disease resistance and banana growth. As shown in as 32.7% c. The N as 32.7% a. Withoup = 0.059). Utilization of BCF and fertilize could significantly increase the P accumulation no matter how the pathogen was inoculated or not. The P accumulation was increased by 54.4% in FBCF treatment in −Foc Tr4 treatment. However, it increased by 44.8% in +Foc Tr4 one . The fertilizer combined with BCF could improve the K accumulation in banana no matter how the pathogen was inoculated or not. In −Foc Tr4 treatment, the K accumulation was increased by 55.9% with the application of the BCF. However, in +Foc Tr4 treatment, the K accumulation was raised by 40.7% in FBCF , peroxidase (POD) and catalase (CAT) are recognized to be the main enzymes of the plant antioxidant system. Cheng et al. (2020) and Dvorak et al. (2021) found that the reactive oxygen metabolic balance was able to be maintained by resisting and eliminating reactive oxygen and oxygen free radicals through increase in POD, CAT and SOD activities in plant when the plant was infected Foc Tr4 pathogen. Thus, the damage of reactive oxygen species to membrane was prevented and integrity and stability of the membrane system were maintained. Finally, pathogen invasion and reproduction were stopped, while plant resistance was enhanced ,50. Lin Banana wilt would be observed when the root of the plant was infected by the pathogen and the pathogen expanded to pseudostems . The sim(1)The combination application of alkaline fertilizer and biocontrol fungus reduces incidence of yellow leaves, rhizome browning degree, and banana Fusarium wilt incidence significantly.(2)The synergistic effects of alkaline fertilizer and biocontrol fungus increase root activity and root antioxidant enzyme activity of banana plants remarkably. Meanwhile, the absorption and accumulation of nitrogen, phosphorus and potassium nutrients in banana plants are boosted, so as to improve the growth of banana and enhance the plant to resist the Fusarium wilt disease.(3)Synchronized application of alkaline fertilizer and biocontrol fungi reduces the Foc Tr4 pathogen infection and colonization in banana root and spreading to rhizome and pseudostem. Tylosis formation is then prevented and thus the vascular vessel blockage is avoided to ensure the normal transport of water and nutrients between underground parts to aboveground ones.