The low mass and high specific stiffness of Mg alloys make them particularly interesting as means of transportation. Due to further desirable properties, such as good machinability and excellent castability, Mg alloys have gained acceptance as castings in high-volume applications, such as gearbox housings and automotive steering wheels. However, in forming processes, such as extrusion and forging, Mg alloys find little to no industrial use at the moment. The reasons for this are their poor formability, which is reflected in limited processing speeds and low ductility, and their modest mechanical performance, compared to competing materials, such as Al alloys and high-strength steels. Much research is being conducted worldwide on high-strength Mg alloys, most of which rely on high levels of rare earths, making these materials both ecologically and economically questionable. Here, it is shown that high yield strengths (>300 MPa) can be achieved in the Mg–Al–Ca system while maintaining good ductility, using only low-cost elements. The investigations have shown that these properties can be adjusted over broad alloy compositions, which greatly simplifies both the processing and recyclability. Magnesium alloys can be very beneficial for automotive as well as aviation applications, where low weight of parts is crucial. While the casting of magnesium, especially high-pressure die casting, is widely used in the industry, wrought applications are still scarce. The reasons for this are the typical higher costs for extruded profiles and lower mechanical properties, when compared to aluminum. Nevertheless, while the mechanical properties achieved in casting processes are limited, high strengths can be achieved via extrusion.High-strength magnesium extrusion alloys typically rely on at least one of the two main strengthening mechanisms: grain refinement, and/or precipitation hardening. Grain refinement is especially effective in Mg alloys, as the Hall–Petch constant is generally higher when compared to Al alloys . As the When investigating the works done on high strength Mg alloys, two major principal alloying groups can be identified: rare earth element (REE) containing alloys, and REE free materials. In the case of the alloys using REE, the achieved strengths are often dependent on the rare earth content of the alloy. The highest strengths are, therefore, achievable with high amounts of REE . BecauseWhile there have been multiple REE free alloying systems with high strength investigated ,7, the sThis system uses cheap and abundant alloying elements and offers a multitude of processing possibilities. Aluminum is the most important alloying element for Mg, both for casting- and wrought alloys, as it increases the castability and mechanical strength and enables the ability of precipitation hardening ,9. CalciMatching the already described hardening mechanisms (precipitation hardening and grain refinement), two contrary processing trends for Mg–Al–Ca alloys can be found within the literature. One procedure uses the sequence of homogenization, forming and subsequent heat treatment steps, as known from age hardenable Al alloys, aiming to achieve high strengths in the final heat treatment (T6-temper) . Since mThe other processing scheme is mainly based on the formation of small grains and the related increase in mechanical properties, such as hardness and ductility. In this case, heat treatments are not necessarily required, which reduces the processing steps and the associated disadvantages  accordingly. Nevertheless, as the grain size must be minimized for the improvement of the mechanical properties, a high degree of deformation has to be achieved in a material with low grain growth tendencies. This is usually done at low extrusion temperatures and reduced forming speeds, demonstrating this principle for various alloying systems ,26. In tAs can be seen in For this behavior, we investigated a wide range of Mg–Al–Ca–Mn alloys processed with identical processing parameters to improve comparability, focusing on alloys with an Ca/Al ratio of ~1 with the sum of Al+Ca content ranging from 1 to 10 wt.%. All alloys shown were processed by indirect extrusion at 350 °C. Both microstructural and mechanical properties were analyzed, and the results shown should help to further promote understanding of this interesting alloying system.® T20/H, Lilienthal, Germany) in a mild steel crucible with a melt capacity of 30 kg, using commercially pure Mg (99.8%) and Al (99.85%), Ca granules (99.9%) and MnCl2 flakes (98%). Ar with 1 vol.% SF6 was used as a cover gas during the melting and handling.A variation of Mg–Al–Ca–Mn alloys with a Ca/Al ratio of ~1 and a total Al+Ca content of ~1 wt.% to ~10 wt.% was used for the studies. The nominal chemical compositions of the alloys are given in The alloys were gravity cast into cylindrical billets with a diameter of 65 mm and a height of 240 mm in a tempered (approx. 150 °C) mild steel mold. The billets were then turned to a diameter of 48.5 mm and a length of 180 mm for subsequent extrusion. No homogenization heat treatment was performed prior to extrusion.The billets were then inductively heated and indirect hot extruded, using a 1.5 MN direct extrusion press  to circular profiles with a diameter of 10 mm at 350 °C with an extrusion ratio of 25:1. The ram speed was 0.45 mm/s, which resulted in a die exit speed of 0.70 m/min. The profiles were left on the conveyor of the extrusion press until fully cooled. No additional heat treatment was performed.After extrusion, tensile test samples acc. to DIN 50125 A8 × 40 were manufactured out of the profiles, and at least five samples each were tested at room temperature, using a Zwick Z100 tensile testing machine . Microstructural investigations were carried out using optical light microscopy and scanning electron microscopy (SEM). A Tescan MIRA3 field emission gun (FEG) scanning electron microscope  operated at 20 kV and a working distance of 15 mm was used for imaging, energy dispersive X-ray spectroscopy (EDS) and electron backscattered diffraction (EBSD). The images were taken using a 4-quadrant solid-state backscattered electron (BSE) detector. The element analysis was performed with an EDAX  Octane Elect silicon drift detector system, and an electron backscattered diffraction (EBSD) was performed using an EDAX Hikari Plus EBSD camera. EBSD data analysis was carried out with EDAX OIM Analysis 8 . For cleanup, we used one iteration of grain dilatation with a grain tolerance angle of 5° and a minimum grain size of 2 px and neighbor CI correlation with a min. CI of 0.1. For the grain size analysis, a misorientation angle of 15° was used to determine the grain boundaries. For a measurement of the recrystallized grain fraction, the recrystallized and non-recrystallized grains were divided by their grain orientation spread (GOS) as discussed by .Thermodynamic calculations were made using Thermo-Calc software (Version 2021b) and databases TCMG5 and TCMG6 . The calculations were performed, using average values of the compositions given in To provide an in-depth analysis of the produced alloys, microstructural investigations using light microscopy as well as SEM/EBSD were performed, and CALPHAD calculations were used to obtain additional information on the phase formation. The mechanical properties were evaluated by tensile tests at room temperature. To allow for a better understanding of the phase formation in the alloys used in this work, the respective thermodynamic calculations in equilibrium state are shown in 2Ca, which forms either directly from the liquid or precipitates from the supersaturated solid. In ternary Mg–Ca–Al alloys, Al2Ca, 2Ca and Mg2Ca can form, depending on the local Ca/Al ratio [In binary Mg–Ca alloys, the prevalent phase is the C14-structured MgAl ratio ,34,35.8Mn5 during solidification before reactions between Ca and Al occur, the effective Ca/Al ratio increases as Al is consumed. This effect is more pronounced in alloys with a lower Ca+Al content, when compared with the higher alloyed ones.All investigated alloys possess a Ca/Al ratio of ~1 (in wt.%) but differ in the total amount of Ca+Al. Since the Mn present in the alloys forms Al8Mn5 is formed before α-Mg, while in AX01 and AX11, the temperature of formation is lower than the formation of α-Mg. As the Al8Mn5 phases form directly from the melt, the solidification speed is critical to the size of the Al–Mn particles, especially as they cannot be dissolved during the subsequent processing steps.In the alloys with a higher Mn content , Al2Ca phase forms directly out of the melt at eutectic temperature. With reduced alloying content, the phase formation is shifted toward lower temperatures. Therefore, in AX11, it precipitates from the supersaturated solid, while in AX01, the C36 phase does not form. On the other hand, the temperatures of phase formation for C14 (Mg2Ca) and C15 (Al2Ca) are independent of the alloying content, except for AX01 . In all alloys, except for AX01 and AX11, the C36 for AX01 a.2Ca is present in the equilibrium state and only Mg2Ca, Al2Ca and Al8Mn5 can be found in addition to the Mg solid solution (At extrusion temperature (350 °C), the calculations indicate that no solution f. NevertIt should be noted that, under real casting conditions, in contrast to the equilibrium calculations shown, phase formation during solidification may be shifted toward lower temperatures, due to the local solidification rate. This is particularly noteworthy because the materials in this study were extruded without prior heat treatment, and thus a non-equilibrium state has to be assumed. In this case, phases can additionally settle out of the supersaturated matrix during heating/extrusion.In the as-cast microstructure , lamellaThe grain size of the as-cast materials is given in The pole figures depicted in The mechanical properties of the as-extruded materials obtained from tensile tests performed at room temperature are given in The yield strength as well as the ultimate tensile strength increase continuously with increasing the content of Ca+Al up to AX43, which shows the highest strength of all investigated alloys. The elongation at break decreases with increasing the alloying content, where AX43 possesses the lowest value. For higher alloying contents, these trends reverse, and the strength decreases while the elongation is slightly improved, compared to AX43. Therefore, AX55, although it contains the highest amounts of Ca+Al, has strength values comparable to AX22 but with a lower elongation at break.To assess the thermal stability of the alloys, AX11 was investigated by annealing treatments, reflecting potential applications. AX11 was chosen based on its overall good combination of strength and elongation at break. Additionally, the relatively low combined content of Al+Ca might lead to increased temperature response and, therefore, accelerated grain growth. The extruded material was exposed to temperatures of 150 °C and 250 °C for 9 and 24 h, respectively. The results of the tensile tests can be seen in As shown in The microstructure found in the as-extruded material is typical for this kind of alloy and is described in detail by . The finThe measured texture is common for as-extruded Mg–Al–Ca alloys, i.e., a basal fiber texture, as reported in . The texThere are multiple reasons for the good mechanical properties of this alloy system. Regardless of the initial cast grain size, a fine microstructure is obtained after forming. These small grain sizes within the DRXed areas provide good ductility as well as high strengths by grain boundary hardening . Additionally, the nDRXed grains of this bi-modal structure are an important feature, which can directly influence the resulting YS by sub-grain hardening, as reported in . AdditioAs aging treatments show little effect on the mechanical properties of this type of as-extruded Mg–Al–Ca–Mn alloy ,31, compBy maintaining a constant Ca/Al ratio, a wide selection of alloys with similar structure and attractive mechanical properties  can be created.The combination of extrusion process and intermetallic phase formation results in a bi-modal grain structure with a large fraction (up to 89%) of fine recrystallized grains (<2 µm). The intermetallic phases enable this fine grain structure by inhibiting grain growth and facilitating recrystallization.Alloy production can be realized with the use of abundant raw materials .No heat treatments are necessary during processing.The obtained mechanical properties are well maintained after exposure for 24 h at elevated temperatures (250 °C), as the intermetallic phases present are thermally stable.No narrow tolerances for chemical composition (except a Ca/Al ratio of >0.9) need to be maintained; therefore, alloy production and recycling are simplified.We investigated the microstructure and mechanical properties of extruded Mg–Al–Ca–Mn alloys with a Ca/Al ratio of ~1 (in wt.%) and a total Ca+Al content ranging from ~1 wt.% up to 10 wt.%, and achieved the following results:Concluding, we believe that this advantageous combination of properties could arouse industrial interest and enable future applications of Mg–Al–Ca–Mn alloys.


Fusarium oxysporum or the foliar pathogen Botrytis cinerea in tomato, the consortia showed an extended functionality, effectively controlling both pathogens under any of the application schemes, always reaching the same protection levels as the best performing single strains. Our findings illustrate the potential of microbial consortia, composed of carefully selected and compatible beneficial microorganisms, including bacteria and fungi, for the development of stable and versatile biological control products for plant protection against a wider range of diseases.The use of beneficial microorganisms for the biological control of plant diseases and pests has emerged as a viable alternative to chemical pesticides in agriculture. Traditionally, microbe-based biocontrol strategies for crop protection relied on the application of single microorganisms. However, the design of microbial consortia for improving the reliability of current biological control practices is now a major trend in biotechnology, and it is already being exploited commercially in the context of sustainable agriculture. In the present study, exploiting the microbial library of the biocontrol company Koppert Biological Systems, we designed microbial consortia composed of carefully selected, well-characterized beneficial bacteria and fungi displaying diverse biocontrol modes of action. We compared their ability to control shoot and root pathogens when applied separately or in combination as microbial consortia, and across different application strategies that imply direct microbial antagonism or induced systemic plant resistance. We hypothesized that consortia will be more versatile than the single strains, displaying an extended functionality, as they will be able to control a wider range of plant diseases through diverse mechanisms and application methods. Our results confirmed our hypothesis, revealing that while different individual microorganisms were the most effective in controlling the root pathogen  A plethora of soil-borne microorganisms live associated with plant roots, and although some are detrimental, others provide important benefits to the host plant, from improved nutrition through growth and protection against multiple abiotic and biotic stresses . NowadayThe ability of microorganisms to control pests and diseases has been well documented, but the variability of results often recorded under field conditions is one of the major challenges for wider adoption in agriculture . OriginaPlant microbiome engineering and the design of synthetic microbial communities (SynComs) to improve crop productivity and resilience is a major research topic in this decade . SynComsTrichoderma spp., and arbuscular mycorrhizal fungi (AMF) have been shown to effectively protect plants against diverse pests and diseases through different mechanisms  those with direct effects on the attacker and (ii) those with indirect, usually plant-mediated effects. Direct effects are mostly based on microbial antagonism through antibiosis, competition for nutrients or colonization niches, and/or parasitism . Indirecchanisms .Bacillus and Pseudomonas , we demonstrate the advantages of targeting microbial consortia as versatile products for efficient biocontrol of diverse plant diseases.In this study, we test the hypothesis that microbial consortia are more versatile than individual microbial inoculants, displaying an extended functionality in the biocontrol of a wider range of plant diseases and application methods through the combination of diverse modes of action. For that, we designed different SynComs by carefully selecting diverse and well-characterized microbial biocontrol agents, including A careful selection of beneficial microorganisms to create synthetic microbial consortia was performed focusing on the main groups of rhizospheric beneficial microorganisms such as PGPR, mycoparasitic fungi, and AMF. An extensive literature review on biocontrol studies of known BCAs was performed, taking also into account as potential candidates the microbial strains available at Koppert Biological Systems. The most relevant studies considered are summarized in Bacillus amyloliquefaciens strains CECT 8238 and CECT 8237, formerly known as Bacillus subtilis UMAF6614 and UMAF6639, respectively  for 24 h at 28°C. After that, a single colony from TSA culture was inoculated in 25 ml of Difco sporulation medium  inoculated with a single bacterial colony from TSA culture and incubated overnight at 28°C with rotary shaking at 200 rpm. After that, 1 ml of pre-culture was inoculated in 25 ml of TSB medium and placed in a rotatory shaker (200 rpm) at 28°C. After 150 min of incubation, with bacterial growth in exponential phase, the cell concentration was calculated measuring the O.D. (620 nm) of the bacterial culture on Shimadzu UVmini-1240 Spectrophotometer. The bacterial culture was centrifuged at 5,000 rpm for 15 min, and after discarding the supernatant, the pellet containing the bacterial cells was resuspended in sterile tap water to a final concentration of 1 × 107 colony forming unit (cfu)/ml.Trichoderma harzianum strains were cultured on potato dextrose agar  for 7 days at room temperature. Spores were collected from sporulating plates in sterile tap water, and the concentration of the spore suspension was quantified using a Bürker-Türk counting chamber and adjusted to 1 × 107 spores/ml.Rhizophagus irregularis was grown in a monoxenic culture on a minimal (M) medium and using Agrobacterium rhizogenes-transformed carrot (Daucus carota) roots as a host root  and placed in a rotary shaker (110 rpm) at room temperature. After 4 days of incubation, the liquid culture was filtered using a sterile miracloth filter, and the spore concentration was quantified using a Bürker-Türk counting chamber. The resulting spore suspension was centrifuged at 9,500 rpm for 15 min and after discarding the supernatant, the pellet containing the spores was resuspended in sterile tap water to a final concentration of 1 × 108 spores/ml.Botrytis cinerea was cultured on PDA at 20°C. Spores were collected from sporulating 14 days old plates in potato dextrose broth , and the concentration of the spore suspension was quantified using a Bürker-Türk counting chamber and adjusted to 1 × 106 spores/ml.Bacillus amyloliquefaciens CECT 8238 and CECT 8237, P. azotoformans, P. chlororaphis, and T. harzianum T22 and ESALQ1306 were initially evaluated in vitro, in confrontation assays against F. oxysporum and B. cinerea. For Trichoderma, one PDA plug (4 mm) of Trichoderma culture and one of the pathogen cultures were placed on PDA plates with 4 cm of distance from each other. For Bacillus and Pseudomonas, 10 μl drop of TSB liquid culture grown overnight was used instead of PDA plugs. As a control, a plug of the pathogen culture was placed in the Petri dish without any antagonist. All treatments were replicated three times. All plates were incubated at 25°C for 7 days. The radius of the pathogen colony in the confrontation plates was measured and compared to the radius of the pathogen colony in the control plates.The antagonistic activity of the individual strains in planta through several bioassays including diverse inoculation methods and targeting different pathogens. This strategy allows testing in vivo different modes of action ranging from direct antagonism to indirect plant-mediated effects. Thus, we tested through seed inoculation suppression of the root pathogen F. oxysporum and ISR against the foliar pathogen B. cinerea, and suppression of B. cinerea by foliar spray application.Biocontrol potential was tested 7 cfu or spores/plant in the seed application and at 1 × 107 cfu or spores/ml in the foliar application. For the AMF treatments, a suspension of 1,000 spores of R. irregularis was applied per plant. Regarding the consortia, the first microbial consortium, SynCom1, was composed of one strain from each genus . The second one, SynCom2, was composed of all selected microorganisms . Both consortia were tested at two doses: A—the same amount of each microorganism in both consortia  or B—same total cfu per consortia .In all bioassays, individual microorganisms and different synthetic consortia were tested . All micSolanum lycopersicum cv. Money maker seeds  were surface-sterilized by immersion in 5% sodium hypochlorite solution for 1 min followed by at least three washing steps in sterile water for 10 min each. The surface sterilized seeds were dried in a laminar flow cabinet and used for the experiments. The growing substrate was gamma-irradiated nutrient poor peat soil . All experiments were performed in a growing chamber at Koppert B.V.  under controlled conditions (25°C:23°C day:night with photoperiod 16 h:8 h light:dark and 60% of relative humidity).6 conidia/g of soil F. oxysporum f.sp. radicis-lycopersici conidia. The F. oxysporum conidia were carefully mixed through the soil by hand. Then, 12 seeds were sown in each container in a regular grid and inoculated with the microbial treatments  were filled with 300 g of soil previously moistened with tap water  and infected with 1 × 10eatments  by pipetB. cinerea conidia suspension (1 × 106 conidia/ml). The leaves were placed in six sealed boxes with high humidity at 20°C, locating one replicate from each treatment in each box. About 60 h after infection, the diameter of the resulting necrotic lesions was measured using a digital caliper.Tomato seeds were sown in pots filled with 250 ml of soil (one seed per pot). Plants were grown for 7 weeks and watered two times per week with water and once per week with Long Ashton nutrient solution . The indR. irregularis was also included, both, individually and in the consortia. A disease control treatment was included where the seeds only received water without any BCA microbial addition. Each treatment was replicated 12 times. We used a randomized complete block design. Plants were watered two times per week with water and once per week with Long Ashton nutrient solution (B. cinerea conidia suspension (1 × 106 conidia/ml). The leaves were placed in 12 sealed boxes with high humidity at 20°C and locating one replicate from each treatment in each box. About 48 h after infection, the diameter of the necrotic lesions was measured using a digital caliper.Tomato seeds were sown in pots containing 250 ml of soil (one seed per pot) and the microbial treatments  were appsolution  but withR. irregularis) was initially inoculated at 1 × 107 cfu/plant, resulting in a total concentration of 4 × 105 cfu/g of soil for each strain (12 plants/300 g of soil). Finally, the seeds were covered with sterile vermiculite to avoid desiccation and undesired contaminations. We included a control treatment without any microbial inoculation. Each treatment was replicated five times. We used a randomized complete block design. Microbial colonization was evaluated 15 days after sowing using methods described in the next section.Rectangular plastic containers of 18 cm × 13 cm × 6 cm (length × width × height) were filled with 300 g of soil previously moistened with tap water . Then, 12 surface-sterilized tomato seeds were sown in each container in a regular grid. The seeds were inoculated with the different microbial treatments  by pipetTrichoderma, we estimated for each genus the number of colony forming units (cfu) per gram of rhizospheric soil. For this, 1 g of rhizospheric soil was sampled, diluted in 9 ml of sterile tap water, and homogenized in a horizontal shaker at 350 rpm for 1 h. Serial dilutions were plated on PDA + igepal (11 ml/L) + tetracycline (50 μg/ml) when targeting Trichoderma and on TSA + natamycin (0.1 g/L) when targeting bacteria. The plates were then incubated at 25°C and cfu were counted after 24 h for bacteria and after 48 h for Trichoderma. In consortia treatments, Bacillus spp., Pseudomonas spp., and Trichoderma spp. were distinguished morphologically, as they are well-characterized strains in the Koppert collection . Model validation was performed graphically by inspecting the residuals and fitted values . The pathogen fully compromised plant survival, as no plants survived in the disease control, while almost 100% survival was found in the absence of the pathogen (non-diseased control)  . None ofeatments .B. cinerea was also tested in planta, applying the BCA treatments by spraying the leaves before B. cinerea infection. Among single microbial treatments, P. chlororaphis, P. azotoformans, and T. harzianum T22 were able to reduce the area of the necrotic lesion caused by B. cinerea by 56, 45, and 38%, respectively, compared to the control treatment  and the SynCom2B  was confirmed in the control treatment plates (R. irregularis) was initially inoculated at a total concentration of 4 × 105 cfu/g of soil for each strain .The repeated success of the consortia across all the t plates . Each miBacillus spp. abundance at the end of the experiment was similar to that initially inoculated both in single strain and SynCom1 treatments   compared to the individual P. azotoformans treatment (around 6.5 × 106 cfu/g of soil) , pointing to a potential positive effect of AMF presence in this consortium  , evidencof soil) . Regardinsortium .Trichoderma spp. abundance in the individual treatments was 9.3 × 105 and 6.3 × 105 cfu/g of soil in T. harzianum T22 and ESALQ1306, respectively, which in the case of T22 is more than double of the concentration inoculated  and around 1.5 × 106 cfu/g of soil in SynCom2 treatments equivalent to the sum of both Trichoderma strains co-inoculated in this consortium (oculated . Regardinsortium .R. irregularis was 1.2% when applied individually (Finally, the percentage of root length colonized by vidually . Root covidually , confirmvidually .In the present study, by combining well-characterized and compatible microorganisms, including bacteria and fungi, we demonstrated the potential of microbial consortia to effectively control fungal pathogens with different lifestyles through direct and plant-mediated disease suppression and using different application methods. Our findings pinpoint the design of synthetic microbial consortia for biocontrol of plant pathogens as a potential strategy to extend the functionality and versatility of microbial biological control.Across the different experiments, different individual microorganisms were the most effective in the different scenarios, depending on the type of pathogen or the strategy used for its control. Remarkably, the consortia effectively controlled all pathogens in all different bioassays, both through direct antagonism by seed or foliar application, or inducing plant systemic resistance against foliar pathogens by seed inoculation (results summarized in Bacillus amyloliquefaciens strains CECT 8238 and CECT 8237 have been shown to promote plant growth and effectively control diverse microbial pathogens through direct antagonism or indirectly through ISR (P. chlororaphis MA342 has been described to effectively control seed and soil pathogens via direct antagonism (P. azotoformans F30A effectively enhance plant emergency and growth (T. harzianum strain T22 is one of the best characterized and commercialized Trichoderma strains. It effectively antagonizes soil pathogens (T. harzianum ESALQ1306 has been shown to highly reduce Sclerotinia sclerotiorum disease severity through parasitism and to induce ISR against spider mites (R. irregularis is not a direct antagonist of plant pathogens, but is able to induce ISR against root and foliar pathogens (T. harzianum, already commercialized as BCA by Koppert Biological Systems all over the world from vegetable and ornamental to field and row crops.For the design of the synthetic microbial consortia, we selected different strains aiming to combine different mechanisms for biocontrol from the production of diverse antimicrobial metabolites through mycoparasitism to ISR. ough ISR . P. chlotagonism  and prottagonism . P. azotd growth  and can d growth . T. harzathogens  and can athogens . Besideser mites . In contathogens . All in via ISR. The SynComs performed consistently well across the different pathosystems. Yet, differences between the SynComs and the individual components were relatively mild in terms of efficacy/degree of control.Most studies focusing on the use of microbial consortia for disease control are looking for synergistic or additive effects, aiming to achieve a higher pest or disease control than their components. While some of these studies have indeed reported positive effects , many otBacillus and Trichoderma performed in the consortia as good as when individually inoculated, and Pseudomonas even benefited from the combination with the other organisms, as they performed better in the SynComs than when inoculated alone. It is important to note that R. irregularis was neither negatively affected in early nor late symbiosis stages by the presence of Trichoderma spp., as demonstrated by the similar mycorrhizal colonization in roots inoculated with the AMF alone or as part of the consortia. This is remarkable, as the compatibility of Trichoderma spp. with mycorrhizal fungi is frequently questioned because of the high mycoparasitic potential of these biocontrol fungi. In fact, Trichoderma is able to parasite AMF in vitro (Trichoderma-AMF synergistic effects have been reported (Microbial compatibility is a key factor when designing a microbial consortium, essential for the successful establishment and functionality of the included microorganisms and the success of SynCom products. In our study, the conservation of the biocontrol effectiveness in the SynComs to the same levels as the best performing individual isolates supported the compatibility between the coexisting microorganisms. We further tested their compatibility in our consortia by assessing the microbial survival in a plant–soil-based experiment, and we did not find any negative interaction between them. Instead, in vitro , but othin vitro . Even moreported . AlthougOverall, our findings highlight the potential multifunctionality of SynComs for biological control. Combining compatible beneficial microorganisms with complementary effects on different targets, direct and indirect mechanisms of control and/or effective under different conditions will lead to the development of biocontrol products with increased versatility. To became commercial products, consistency of the outcomes needs to be tested and finally validated across multiple field trials in the geographical regions where is aimed to be used. This is a key step for the successful application of this sustainable technology in agriculture.The original contributions presented in the study are included in the article/MP, RS, OK, and ZM designed the experiments. ZM performed all the experimental work. OK and ZM analyzed the data. RS and OK critically revised the manuscript. MP and ZM wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Compared to the 1970s, the edge of the Ecology Glacier on King George Island, maritime Antarctica, is positioned more than 500 m inwards, exposing a large area of new terrain to soil-forming processes and periglacial climate for more than 40 years. To gain information on the state of soil formation and its interplay with microbial activity, three hyperskeletic Cryosols (vegetation cover of 0–80%) deglaciated after 1979 in the foreland of the Ecology Glacier and a Cambic Cryosol (vegetation cover of 100%) distal to the lateral moraine deglaciated before 1956 were investigated by combining soil chemical and microbiological methods. In the upper part of all soils, a decrease in soil pH was observed, but only the Cambic Cryosol showed a clear direction of pedogenic and weathering processes, such as initial silicate weathering indicated by a decreasing Chemical Index of Alteration with depth. Differences in the development of these initial soils could be related to different microbial community compositions and vegetation coverage, despite the short distance among them. We observed—decreasing with depth—the highest bacterial abundances and microbial diversity at vegetated sites. Multiple clusters of abundant amplicon sequence variants were found depending on the site-specific characteristics as well as a distinct shift in the microbial community structure towards more similar communities at soil depths > 10 cm. In the foreland of the Ecology Glacier, the main soil-forming processes on a decadal timescale are acidification and accumulation of soil organic carbon and nitrogen, accompanied by changes in microbial abundances, microbial community compositions, and plant coverage, whereas quantifiable silicate weathering and the formation of pedogenic oxides occur on a centennial to a millennial timescale after deglaciation. Considering the particular vulnerability of the Antarctic environment to climate change, studies on soils from glacier forelands could provide indications of how climate changes at the global scale will affect soil formation at the regional scale. By substituting space with time, chronosequences of proglacial environments are an important tool to understand primary succession and soil forming processes5, and were therefore used to study the succession of soil microbial communities and their influence on initial soil formation in the past8. Such microbial populations with different abundances, community structures and diversities are among the first organisms to colonize recently deglaciated areas11. Their activities within biogeochemical cycles such as the fixation of carbon and nitrogen into bioavailable forms7 can promote environmental changes that facilitate the succession of organisms at higher trophic levels14. In order to understand the relationship between primary and secondary succession in proglacial environments and to shed light on the influence of microbial processes on the development of initial soil ecosystems and vice-versa, it is crucial to study the factors that shape the genetic structure of local microbial populations of such environments13.Retreating glaciers in polar and mountainous regions reveal proglacial terrain that is exposed to soil formation and subsequently colonized by microorganisms and plants16, which are well adapted to their harsh environment18. These microbial communities have been described to be influenced by local soil chemical parameters, such as pH19, and soil physical parameters such as grain size distribution and soil moisture20. Also, the microclimate21, vegetation cover, and cryoturbation processes22 play a role in the observed soil properties  in Antarctica. Thus, these properties and processes could have an impact on soil microbial community composition and activity.The present microbial communities in ice-free areas of polar regions are dominated by Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Gemmatimonadetes and Proteobacteria25. Following the regional warming during the last 50 years, a significant loss of ice volume and melting of many outlet glaciers in maritime Antarctica could be observed28. The glacial retreat will probably keep its accelerated pace due to the continuous warming over Antarctica by 0.34 °C per decade29. It will continuously affect soil-forming processes and microbial activity in maritime Antarctica by exposing glacial sediments32, which offers an excellent setting to investigate initial soil-forming processes and the colonization by microbial pioneers before higher plants succeed30. Particularly, the frontal retreat of the Ecology Glacier on King George Island, South Shetland Islands, with approx. 30 m per year since the early nineties opens new terrain for soil-forming processes and terrestrial life33. In a centennial to millennial timescale, the carbon and nitrogen content, as well as the pH are the main soil properties to change on King George Island, leading to the formation of soil horizons34. Similar findings were observed from glacier forelands in Europe, particularly from the Alps in Switzerland38. A recent study in the foreland of the Ecology Glacier on King George Island demonstrated that the diversity and properties of microorganisms in recently deglaciated areas are not only related to age but also to differences in soil stability within the upper centimeters due to the influence of cryoturbation39. Nevertheless, there is still a deficiency of information about the decadal-scale changes of soil properties and their interplay with microorganisms in soil ecosystems in maritime Antarctica.Compared to ice-free areas of continental Antarctica, the soils from maritime Antarctica differ significantly due to higher water availability and warmer temperatures, which lead to deeper active layers and promote vegetative cover and mineral weatheringWe hypothesize that prokaryotic microorganisms initiate/drive soil properties changes  within decades after deglaciation. To test our hypothesis, we related the investigated soil properties to microbial community structure and microbial abundances in the foreland deglaciated after 1979 and compared it with an older soil distal to the lateral moraine (deglaciated before 1956) of the Ecology Glacier. To capture the heterogeneity of the soil landscape, three soils in close proximity (maximum distance of 150 m) formed on the same substrate and in a similar topographic position but with differing vegetation cover were sampled. These soils, which represent a recently deglaciated area, were compared with an older soil that had formed on a similar substrate that had been deglaciated before 1956. We combined grain size and pedochemical analyses with DNA-based molecular biological analyses, including high-throughput sequencing and quantitative PCR, to determine the diversity, distribution, and abundance of microbial communities.40. The rocks exposed after the frontal retreat of the glacier are mainly mafic volcanic rocks from the Arctowski Cove Formation.King George Island is located in the South Shetland Islands archipelago. The stratigraphy of King George Island comprises Upper Cretaceous to Lower Miocene predominantly subaerial volcanic and volcanoclastic rocks. Fossiliferous marine and glaciomarine sediments are more common in the Oligocene to Lower Miocene rocks. Quaternary volcanism along the southern margin of King George Island and the axial part of Bransfield Strait is related to back-arc extension41. Moreover, a usually water-saturated active layer during the summer increases biological and chemical weathering and accumulation of organic carbon42. As a result, a suite of soil-forming processes occurs on the island, such as cryoturbation, gleization, melanization, paludization, and phosphatization. Since most of the soils are relatively young and weakly developed (~ 4000 year BP since the last deglaciation on King George Island)43, these processes are closely linked to the landscape position, parent material and faunal activity (e.g. penguin rookeries). The main resulting soil orders are Cryosols, Leptosols, Cambisols, and Histosols44, but also different soil groups such as Arenosols and Gleysols may occur45. Additionally, Podzols, Umbrisols, Stagnosols and Gleysols were found in the surrounding area of the Arctowski Station on King Georges Island47.The relatively mild and moist conditions in maritime Antarctica compared to continental Antarctica result in frequent freeze–thaw cycles, which foster periglacial processes (e.g. cryoturbation), and chemical and physical weathering48. The margin of the Ecology Glacier was > 500 m inward in 2014 compared with the front line during the late 1970s, where the ice reached the sea  between 1979 and 1989, the speed of deglaciation increased significantly between 1989 and 200149.The study site is located in the foreland of the Ecology Glacier on King George Island characterized by an oceanic polar climate. Temperature measurements recorded by the Chilean Antarctic station President Eduardo Frei Montalva from 1971 to 2004 indicate a mean annual temperature of − 2.3 °C with the coldest temperatures in July and August (mean temperature − 6.5 °C) and the warmest in February (mean temperature 1.6 °C). The mean annual precipitation is < 500 mm, with maximum precipitation during spring/autumn and a minimum during summer/winter39. We sampled three soil profiles  from well-drained positions of lateral moraine deposits with slightly different leeside/windward positions in the present foreland of the Ecology Glacier  is located in a well-drained position distal to the lateral moraine Figs. , 3A on a51. Samples were transported frozen to Germany and stored at a temperature of − 18 °C.Fieldwork was carried out in summer 2014. Soil morphological description followed the guidelines of the Food and Agriculture Organization of the United Nations, and the pedons were classified using the World Reference Base system3) for bulk density were taken from each horizon/depth increment with steel rings in three replicates. Bulk density [g cm−3] was gravimetrically determined including the correction by coarse material52. Bulk samples were air-dried and sieved < 2 mm. The grain size distribution (< 2 mm) of all samples was determined by combined sieving (2000–20 µm) and X-ray granulometry after using 1 M sodium metaphosphate (Na4P2O7) as a dispersant53.Volumetric samples (100 cmt) and soil organic carbon (SOC) were determined by thermal conductivity analysis after heat combustion (1150 °C) with a CNS-element analyzer (Elementar Vario EL III). AMS 14C dating was used to determine the age of the organic carbon of the samples with the highest SOC contents . Soil pH[H2O] and pH[CaCl2] were determined potentiometrically in a 1:2.5 soil to water/0.01 M CaCl2 solution. Pedogenic Fe-(hydr-)oxides (Fed) were extracted by dithionite-citrate-bicarbonate (DCB) solution54. Non- and poorly crystallized compounds of Fe (Feo) and Al (Alo) were extracted by shaking 2.5 g of soil in 100 mL 0.2 M acid ammonium oxalate (pH 3) for 4 h in the dark55. The ratio between total Fe and pedogenic Fe-(hydr-)oxides (Fet/Fed) gives information on the iron release of Fe-bearing minerals, reflecting the intensity of weathering, whereas the ratio Feo/Fed gives information on the degree of iron oxides crystallinity56. Major elements, including Fe (Fet), were measured with a wavelength dispersive XRF device . Prior to preparation, the bulk samples (ratio Li-metaborate to soil 1:5) were ground with an agate mill for 10 min. The Chemical Index of Alteration (CIA) gives information on the ongoing chemical weathering and was calculated according to Nesbitt and Young57. The calculation was as follows [(Al2O3/Al2O3 + Na2O + CaO* + K2O)) × 100], where CaO* represents the amount of silicate-bound CaO.Total nitrogen . In addition, a negative control without any template but the material and chemicals of the extraction kit was included. Sample replicates with very low DNA yields  were sequenced using tagged 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primers58. The used cycler program and reaction mix were described by Meier et al.59. The sequencing was performed on an Illumina HiSeq (2 × 300 bp) by GATC Biotech AG, Germany.Total genomic DNA extracts of each sample as well as an extraction negative control and a positive control . According to the resulting report files, a filtering step was implemented to get fastq sequence identifiers for sequences with a minimum overlap of > 25 bases, maximum mismatches of < 5 bases and no ambigious bases. Next, these sequences were extracted with the filterbyname.sh function from BBTools62 from the raw paired-end fastq file. With QIIME1, sequence orientation was checked and corrected by using the extract_barcodes.py function and the primers were removed using the awk command58. DADA2 was used for filtering, dereplication, chimera check, sequence merge, and amplicon sequence variants (ASV) calling63. The output of DADA2 was taxonomically classified by using QIIME264 and USEARCH65 with SILVA13866. ASVs with a relative abundance of < 0.01% in each sample were excluded from further analysis. For the processing and visualization of the obtained ASV table, R  and PAST467 were used. Sample triplicates were merged by the mean value of their relative abundance before visualization of the sequencing data and before analysis of correlating environmental factors. The hierarchical clustering of the samples using the average linkage method was based on the Bray–Curtis dissimilarity. Demultiplexed raw sequencing data were submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/ena) under the accession number PRJEB37594.The quality of raw sequencing data obtained by Illumina HiSeq (2 × 300 bp) was checked with FastQC68. The used cycler program and reaction mix were described by Meier et al.59.Bacterial abundances were quantified using quantitative PCR (qPCR) and the 314F (5′-CCTACGGGAGGCAGCAG-3′) and 534R (5′-ATTACCGCGGCTGCTGG-3′) primersThe soils KGI A, KGI B, and KGI C did not have properties to differentiate soil horizons and were classified as Hyperskeletic Cryosols. KGI D had distinct soil horizons and was classified as a Cambic Cryosol. Differences in vegetative cover and pedochemical properties were observed between the investigated soil profiles Table .Table 1MH20/pHCaCl2 was found in the uppermost depth increment . With depth, pHH20/pHCaCl2 increased with the highest values in the lowermost depth increment . The vegetation cover was 0%, 5%, 80%, and 100% for KGI A, KGI B, KGI C, and KGI D, respectively. The vegetation cover at KGI B included Usnea antarctica, Deschampsia antarctica, and Colobanthus quitensis. In addition, no substantial accumulation of nitrogen (Nt < 0.03%) nor soil organic carbon (SOC < 0.05%) was observed. KGI C had higher Nt (0.09%) and SOC (1.24%) contents, and its surface was covered with vegetation comprising of Usnea antarctica, Deschampsia antarctica, Colobanthus quitensis, Ochrolechia frigida, and different mosses. The older and well developed soil, KGI D, showed distinct contents of nitrogen , and of soil organic carbon . The complete surface of KGI D was covered with Deschampsia antarctica, Polytrichum spec., Colobanthus quitensis, and Usnea antarctica. 14C dating showed that the humin fraction of soil organic matter in profile KGI C was formed after the melting of the glacier in 1979 ; 3.2% 1957 cal AD ), whereas the humin fraction of the upper 3 cm in profile KGI D is much older )  or vitric (≥ 0.4%) properties. The Feo/Fed and Fet/Fed ratios, and the CIA did not show a clear direction of pedogenic or chemical weathering in KGI A, B, and C. In contrast, freshly formed Fe-(hydr-)oxides were indicated by the Fet/Fed ratio (12.5–12.7) in the upper two horizons of KGI D. The Feo/Fed ratio also shows a higher activity of Fe(hydr-)oxide formation in the upper horizons by a decreasing trend with depth in KGI D. The decreasing CIA with depth (51–49.1) designates initial silicate weathering processes combined with the dissolution of Ca, Na and K bearing minerals.Regarding soil pH, similar trends were observed in the investigated soil profiles. The lowest pHHigh-throughput sequencing resulted in a mean of 577,306 reads per sample, ranging from 12,252 (KGI A 10–20 cm b) to 917,368 (KGI D 15–27 cm b) reads , Chitinophagaceae (3), Chitinophagaceae (4), Candidatus Udaeobacter (2), Sphingomonas (7), Comamonadaceae, Dokdonella, Flavisolibacter, Candidatus Udaeobacter (3), Tychonema CCAP 1459-11B) and was characteristic for the upper depth increments (0–10 cm) of the sites KGI B and KGI C. Cluster 2 was comprised of six ASVs (Sphingomonas (5), Sphingomonas (6), Gaiella (4), Nocardioides (2), Ferruginibacter, Acidiferrobacteraceae (8))) and characteristic for the upper depth increments (0–10 cm) of the site KGI A. Cluster 3 was comprised of six ASVs , Bradyrhizobium, Gemmatimonadaceae (6), Chloroflexi KD4-96, Rhizobiales) and characteristic for the upper depth increments (0–10 cm) of site KGI D. The five ASVs associated within cluster 4 (Gemmatimonadaceae (5), Polaromonas, Chitinophagaceae, Candidatus Udaeobacter, Pseudarthrobacter) were not specific for any depth. Cluster 5 was comprised of 32 different ASVs , Sphingomonas, Sphingomonas (2), Acidiferrobacteraceae, Microtrichales, Acidiferrobacteraceae (2), Sphingomonas (3), Nocardioides, Acidiferrobacteraceae (3), Gaiella, Gaiella (2), Oryzihumus, Acidiferrobacteraceae (4), Acidiferrobacteraceae (5), Gaiellales, Holophagae Subgroup 7, Lysobacter, Gaiella (3), Gemmatimonadaceae (3), Acidiferrobacteraceae (6), Nitrosococcaceae wb1-P19, Nitrosococcaceae wb1-P19 (2), Gemmatimonadaceae (4), Sphingomonas (4), Acidiferrobacteraceae (7), Rhodoferax) and was mainly connected to the lower depth increments (10/15–40/80 cm) of all sites.4 and 109 copies g−1 soil (Table 8 copies g−1 soil), KGI B (1.3 × 109 copies g−1 soil), KGI C (1.8 × 1010 copies g−1 soil), and KGI D (7.3 × 109 copies g−1 soil) had the highest abundances in the uppermost soil layer. With depth, a substantial decrease in abundances was observed, resulting in the lowest abundances in KGI B (2.1 × 106 copies g−1 soil), KGI C (3.8 × 107 copies g−1 soil), and KGI D (6.3 × 107 copies g−1 soil) in the lowermost soil layer. In KGI A, the lowest abundances with 1.74 × 104 copies g−1 soil were found in a depth of 10–20 cm, before increasing to 1.1 × 107 in the lowermost soil layer.Bacterial abundances determined by the quantification of the 16S rRNA gene showed similar trends across all profiles and varied in general between 10il Table . KGI A  as well as a cluster of ubiquitous ASVs (Cluster 4) were observed. The most abundant ASVs in Cluster 4 were metabolically flexible organisms, such as ASVs related to Chitinophagaceae or Polaromonas. Several Chitinophagaceae-related ASVs were present in the investigated soils and especially abundant in the upper depth increments. Chitinophagaceae have been observed in Antarctic soils before73 and were described to play a role in the degradation of chitin and other soil organic compounds74. The degradation of organic compounds in the course of microbial respiration by Chitinophagaceae and other heterotrophic microorganisms could affect the soil pH and might enhance the chemical weathering close to the surface.In accordance with observations in other Antarctic habitatsPolaromonas-associated ASV could be observed in all soils and depths (Cluster 4). These globally occurring organisms are able to survive in a dormant state75 and are, due to high levels of horizontal gene transfer, metabolically versatile76. These organisms are known to utilize a wide range of substrates such as H277 or diverse organic compounds provided for instance by sea spray such as acetate, chloroacetate or octane78 and could be therefore a pioneer species in the recently exposed soil substrates in the foreland of the Ecology Glacier. In contrast to this cluster of frequently occurring ASVs, three different clusters consisted of abundant ASVs were found in the uppermost depth increments of the bare soil , slightly to moderately vegetated soil , and fully vegetated soil . For instance, one important group within cluster 3 are ASVs related to Rhizobiales or Bradyrhizobium, which are known to be associated with the rhizosphere of plants, were particularly abundant in the fully vegetated site KGI D. These organisms are keyplayers for the fixation of nitrogen in soil ecosystems.Additionally, a 10. These communities are dominated by photosynthetic, and heterotrophic N2-fixing bacteria30, resulting in an initial accumulation of labile carbon and nitrogen pools and play therefore an important role as pioneers for the further development of the fresh glacier forefield sediments. However, our dataset showed only low abundances of prokaryotic organisms most probably associated with phototrophic carbon fixation, such as the Cyanobacteria-related Tychonema. Low abundances of Cyanobacteria in recently deglaciated areas are not an uncommon observation in Antarctic soil environments79. In addition to the low abundances of Cyanobacteria, other bacterial groups might be involved in phototrophic carbon fixation, such as Chloroflexi80. Moreover, based on the on the amount of chloroplast-related sequences in our dataset, this process is at least partly facilitated by eukaryotic organisms such as algae in the early stage of soil development. Those microbial pioneers contribute to the stabilization and physical and chemical development of recently exposed substrates82, and initiate a cascade of crucial processes (e.g. carbon and nitrogen accumulation or bioweathering) that result in the formation of soils in which complex vegetation can grow83.The differences in microbial community composition of the four study sites were also reflected in the microbial diversity of the near-surface depth increments, which increased slightly with vegetation coverage. Before lichens or vascular plants appear, abundant and diverse microbial communities are known to colonize recently exposed substrates82. The site-specific microbial communities and the occurrence of defined clusters in the upper part of the soil profiles changed according to the vegetation coverage and potentially related soil properties such as the SOC or the soil pH.As mentioned above, microbial pioneer communities play an important role in initial soil formation. They alter the original soil environment; and are, in turn, influenced by ongoing pedogenic processes, succession, and plant colonization85, by plant litter input34 or by altering thermal and moisture retention of the soil22. To what extent the microbial communities in Antarctic soils are directly influenced by vegetation and vice versa is debated controversially88. As vegetation coverage increased, microbial communities shifted towards plant-related microorganisms in the foreland of the Damma Glacier in the Alps11. However, we could not observe similar effects on the microbial communities in the lower part of the investigated soils in the foreland of the Ecology Glacier. This is probably due to the lack of deeper roots of pioneer plants and the short time since plants colonized the foreland. The effect of plants on microbial community composition seemed to be limited to the upper part of the soils in the foreland of the Ecology Glacier since communities in depths > 10 cm were similar in all soil profiles regardless of plant coverage. Since more developed soils in the ice-free areas of Antarctica did not show mycorrhization, Boy et al.34 concluded that plants influence the colonized soil more by litter input than by direct transfer of photoassimilates to the surrounding soil. The input of plant litter leads to an increase of soil nitrogen and SOC contents especially in the upper centimeters of the soil. The succeeding decomposition of organic compounds in the course of microbial respiration could lead to a decrease of the pH value in the soil. In the upper and even in the lower part of the investigated soils, soil pH was altered by plant coverage, but shows only little influence on microbial community structure. In soil environments, pH usually is a significant attribute that shapes the present microbial community in favor of certain bacterial phyla89. Our results show that in the foreland of the Ecology Glacier, other vegetation-related properties, such as the SOC content, or soil moisture, and thermal retention, might influence the microbial communities close to the surface, not the pH value of the soils.Vegetation can influence the surrounding soil and its properties as well as the present soil microbiome in different ways, e.g. by releasing plant root exudates16. After the initial accumulation of labile carbon and nitrogen pools by microorganisms and the subsequent colonization of plants, the input of additional soil organic matter in the form of litter might sustain a richer heterotrophic community in the otherwise nutrient-poor environments of Antarctica. The presence of vegetation has been suggested to enhance the soil moisture and thermal retention of soils, thus reducing the severity of Antarctic conditions on the soil environment22. The soil moisture affects enzymatic and microbial activity90, the primary production91, and ultimately influences the microbial community structure in a variety of Antarctic habitats92. Another study showed that soil temperature affects microbial community composition and soil respiration93. In addition to the above-mentioned effects of plant colonization on SOC or soil pH, slightly higher and more stable moisture and temperature regimes due to the vegetation-related retention could lead to the differences in community compositions observed in the foreland of the Ecology Glacier, such as increased abundances of Verrucomicrobia-related species.The SOC content has been shown to have a significant influence on microbial communities in cold habitats94 compared several soil profiles in a forested montane watershed, where the most variable communities were located down to a depth of 10 cm and where less diverse and more similar microbial communities could be observed at depths > 10 cm regardless of the landscape position. They suggested that changes in soil properties with depth  represent an ecological filter which makes it difficult for adapted surface-dwelling microorganisms to thrive, and causes a shift in the community composition in deeper soil horizons. Furthermore, changes in soil microstructure, induced e.g. by frequent freeze–thaw cycles and associated changes in pore spacing and nutrient contents have been related to shifts in microbial community compositions in soils from maritime Antarctica59. Meier et al. observed a change towards a lenticular microstructure below 20 cm depth, which was related to significant changes in the microbial community compositions. Some of the observed ASVs in deeper soils were Acidiferrobacteraceae-related organisms, which usually are associated with autotrophic lifestyles such as sulfur and iron oxidation, and have a broad range of possible substrates, such as ferrous iron, thiosulfate or ferric iron95. In initial soils on James Ross Island, Meier et al.59 found similar OTUs in the lower depth increments and connected those to mineral weathering in the course of microbial iron cycling. Cryoturbation, a process that would mix topsoil material with deeper soil horizons and vice versa, was reported to be influential for both abundance and diversity of bacterial communities in the foreland of the Ecology Glacier39. However, our results indicate that cryoturbation in these soils is of minor importance since in all soil horizons and at all study sites a clear differentiation with regard to the community structure with depth was evident.The present microbiome was influenced by the soil properties of the upper centimeters, such as the initial accumulation of SOC and nitrogen and the ongoing soil formation with its initial weathering processes and plant colonization. Conversely, the microbiome in deeper parts of the soil was affected by a variety of soil chemical parameters that change with depth , which explained a significant fraction of changes in the composition of the microbial community in the investigated soils and resulted in different, less abundant, and less diverse microbial communities. Eilers et al.96. Although the investigated areas and soils are ice-free for just a few decades, the bacterial abundances were high (103–1010 copies g−1 soil) showing similar trends across all investigated soils. Grzesiak et al.97 reported > 1010 counts per gram soil for the foreland of the Ecology Glacier. These high bacterial abundances are comparable to abundances observed in other parts of the Antarctic Peninsula59. A positive relationship between microbial abundances and vegetation as well as vegetation-related environmental factors  was also observed by Yergeau et al.86. With increasing soil development along glacier forelands, defined by increasing carbon and nitrogen contents, decreasing pH, increasing vegetation coverage and increasing weathering ratios, we observed increasing microbial abundances which is consistent with other observations in cold environments10. Nevertheless, the relatively high abundances in the upper centimeters could also be influenced by algae and lichens such as Usnea antarctica and its chloroplasts.Depth and soil properties influenced not only microbial diversity and community composition but also microbial abundances, which increased with vegetation cover and decreased substantially with soil depth. Exponentially decreasing microbial abundances and biomass with depth are a common observation in soil environmentst/Fed and CIA) did not show a clear depth differentiation of pedogenic or weathering processes in the recently exposed soils . Therefore, the chemical properties of the parent material remain almost unaltered.The results show that on a decadal timescale after deglaciation, changes in microbial abundances, community compositions, and plant coverage are accompanied by lowering of the soil pH, and initial accumulation of SOC and nitrogen, which are the main soil-forming processes in the soils in the foreland of the Ecology Glacier. In contrast to these rather rapidly changing parameters, the quantifiable formation of pedogenic oxides and the increase in chemical weathering require much more time under the current climatic conditions of King George Island. The initial chemical weathering processes only became evident in the Cambic Cryosol of KGI D, which deglaciated before 1956. The main indication is the formation of Fe-(hydr-)oxides and a slight increase of the CIA at KGI D. On the other hand, the weathering related indices . In the upper depth increments, differences in the soil chemical and microbiological properties were found even between the three sites in the foreland , which became ice-free at the same time. Soil pH and SOC depended on the vegetation coverage of the respective site and especially the soil pH in the vegetated sites could be impacted by microbial degradation of plant litter. The lowering of the soil pH in the bare soil, however, may be explained by more active Chitinophagaceae and other potential heterotrophs, and the degradation of organic material of microbial origin, such as chitin from fungi.t content, or additionally of the Fed/Fet ratio and the CIA at the oldest site KGI D.Soil depth represents a variety of changes in the environment such as the increase in soil pH or the decrease in organic carbon contents and was the strongest determining factor explaining the decrease in microbial diversity and abundances. The microbial communities were similar at all sites in > 10 cm, regardless of their exposure age after deglaciation. This means that cryoturbation processes may not have played a major role since the deglaciation, otherwise we would not have obtained clear depth functions of soil properties such as SOC and NOn a decadal timescale after deglaciation, changes in soil pH, and initial accumulation of soil carbon and nitrogen were the main soil-forming processes, which were accompanied by changes in microbial abundances, community compositions, and plant cover. On a centennial to a millennial timescale after deglaciation, quantifiable silicate weathering and formation of pedogenic (hydr-)oxides could be observed. The cold climate of Antarctica slows down microbial weathering processes and soil formation rates on recently exposed sediments. However, we conclude that prokaryotic microorganisms initiate measurable changes of soil properties such as pH at a very early stage (within decades) before the soil surface is colonized by pioneer plants or soil horizons other than C horizons are detectable, and thereby promote weathering processes. To further verify our conclusions and illuminate the microbial processes driving soil formation, in future studies multiple comparable setups  could be studied and include metagenomic and metatranscriptomic analyses.Supplementary Tables.Supplementary Table S5.


A pulmonary arteriovenous malformation is an abnormal dilated blood vessel that makes direct communication between a pulmonary artery and pulmonary vein and can be associated with hypoxemia or neurological complications, including brain abscess and cerebral infarction. Treatment of pulmonary arteriovenous malformation includes surgical resection and transcatheter embolotherapy, however the adaptation of therapies should be considered when a patient is in bad condition.A 51-year-old man was admitted after developing fever, consciousness disorder, and hypoxemia. Magnetic resonance imaging of the brain showed a brain abscess. Bilateral pulmonary arteriovenous malformations were found by contrast computed tomography. Because of a family history of pulmonary arteriovenous malformation, a history of epistaxis, and the existence of oral mucosa telangiectasia, he was diagnosed with hereditary hemorrhagic telangiectasia and brain abscess caused by intrapulmonary right-to-left shunt. The brain abscess improved with antibiotic treatment; however, the administration of oxygen did not ameliorate his hypoxemia. His hypoxemia was exacerbated by positive pressure ventilation. Considering his systemic and respiratory condition, we considered surgery to involve a high degree of risk. After controlling his brain abscess and pneumonia, transcatheter embolotherapy was performed. This improved his systemic condition, enabling surgical treatment.This middle-aged patient suffering from brain abscess and severe hypoxemia with multiple pulmonary arteriovenous malformations was successfully treated by a combination of transcatheter embolotherapy and surgery. The adaptation and combination of therapies, as well as the sequence of treatments, should be considered depending on the patient status and lesions. A pulmonary arteriovenous malformation (PAVM) is an abnormal dilated blood vessel that makes direct communication between a pulmonary artery and pulmonary vein. PAVMs are mainly found in patients with hereditary hemorrhagic telangiectasis (HHT) . BecauseA 51-year-old man presented to the emergency department of our institution with fever, consciousness disorder, and hypoxemia. Magnetic resonance imaging of the brain showed multiple ring-enhancing lesions, which suggested multiple brain abscesses Fig. . The tra2O and PEEP at 6 cmH2O, breathing 100% oxygen, an arterial blood gas analysis revealed a partial pressure of arterial oxygen (PaO2) of 48.8 mmHg. However, changing the ventilator settings (driving pressure at 10 cmH2O and PEEP at 0 cmH2O) improved his oxygenation: PaO2 of 62.8 mmHg while breathing 100% oxygen. We suspected this was because positive pressure ventilation exacerbated the intrapulmonary right-to-left shunt.Thanks to the intravenous administration of antibiotics, the brain abscess resolved, and the neurological symptoms improved; however, his hypoxemia was not ameliorated. Because pneumonia also occurred during the treatment of the brain abscess, the patient was intubated and an artificial respirator was used. Despite positive pressure ventilation, his hypoxemia worsened. During pressure-controlled ventilation with driving pressure at 14 cmH2 of 58.7 mmHg while breathing 70% oxygen. In order to improve his hypoxemia and prevent a relapse of the neurological complications, we planned to treat his PAVMs. We considered that surgical treatment was suitable for the large PAVM in the right S3, due to the safety and certainty of therapy; however, the difficulty in maintaining stable breathing during positive pressure and one-lung ventilation initially prevented us from selecting surgical treatment. Because of the control of the patient’s infectious diseases, transcatheter embolotherapy was performed to treat the left PAVM.Even after the improvement of pneumonia with the use of additional antibiotics and ventilator weaning, an arterial blood gas analysis revealed PaO2 had improved to 82.7 mmHg while breathing room air. The patient was able to undergo rehabilitation.The occlusive coils embolized the left PAVM, which was located in S10, resulting in a slight improvement of the patient’s hypoxemia. After embolizing the largest PAVM on the right, which was located at S3, the patient’s oxygenation improved (Fig. Surgical treatment was performed under one-lung ventilation in the left lateral decubitus position. A 20-cm anterolateral skin incision was made along the fourth intercostal space. We confirmed the PAVMs of S1 and S3 and performed right upper lobectomy. In surgery, we found a coil was present in V3 after dissection of the mediastinal pleura and connective tissue. We therefore transected V1, V2, and V3, to avoid cutting or releasing the coil (Fig. 2 level while breathing 100% oxygen increased up to 452 mmHg, and his dyspnea was ameliorated. Using pulmonary shunt fraction measurement with the 100% oxygen method, the shunt fraction was found to have improved from 27.9 to 11.8%. He was discharged to home on the 37th postoperative day after rehabilitation.Transcatheter embolotherapy and surgical treatment were uncomplicated. The patient’s PaOPatients with PAVM can present hypoxemia due to anatomic right-to-left shunt. Since PAVMs may cause life-threatening complications such as ischemic strokes, cerebral or peripheral abscesses, hemoptysis, and hemothorax, PAVMs of any size should be considered treatment regardless of its size and complications . BecauseTreatment for PAVM includes surgical resection and transcatheter embolotherapy. We successfully treated a patient with bilateral PAVMs in bad condition using a combination of these treatments. Appropriate treatments should be selected depending on the number and size of lesions and the patient’s physical status.


In dairy cattle herds milked by automatic systems, the absence of a human milker originates the need for control systems to monitor the milking process and cow conditions. Modern milking robots are equipped with a lot of sensors that, at each milking (2.5–3 times a day), record data on milk yield and quality, milking efficiency, cow welfare, and health with particular focus to udder conditions. Mastitis is one of the most frequent and serious diseases of dairy cow that negatively affects milk quality and yield, reduces animal welfare, and often implies the use of antimicrobial drugs. At the moment, the alerting systems for mastitis risk is generally based on monitoring milk electrical conductivity, color, and/or temperature, but these indicators have limited reliability. Other information gathered by automatic sensors, already implemented in commercial robots, could be useful to early detect mastitis. Using a multivariate approach, our study showed that the deviations over time of milk electrical conductivity, milk yield, and milk flow of single quarters in comparison with the whole udder are potential indicators, alone or in combination, for altered udder conditions. The results could be useful for the development of new algorithms more effective in the early detection of mastitis.Automatic Milking Systems (AMS) record a lot of information, at udder and quarter level, which can be useful for improving the early detection of altered udder health conditions. A total of 752,000 records from 1003 lactating cows milked with two types of AMS in four farms were processed with the aim of identifying new indicators, starting from the variables provided by the AMS, useful to predict the risk of high milk somatic cell count (SCC). Considering the temporal pattern, the quarter vs. udder percentage difference in milk electrical conductivity showed an increase in the fourteen days preceding an official milk control higher than 300,000 SCC/mL. Similarly, deviations over time in quarter vs. udder milk yield, average milk flow, and milking time emerged as potential indicators for high SCC. The Logistic Analysis showed that Milk Production Rate (kg/h) and the within-cow within-milking percentage variations of single quarter vs. udder milk electrical conductivity, milk yield, and average milk flow are all risk factors for high milk SCC. The result suggests that these variables, alone or in combination, and their progression over time could be used to improve the early prediction of risk situations for udder health in AMS milked herds. Automated Milking Systems (AMS) are spreading rapidly in dairy cattle farms; at the beginning of the 2000s there were about 1250 milking robots around the world [The spread of AMS and the parallel reduction in human presence have generated the need to incorporate into the system automatic sensors for monitoring milk production, milking efficiency, and animal conditions. AMS is integrated with a variety of sensing equipment for the detection of multiple indicators during the milking process . In recent years, a number of sensors has been developed for monitoring additional variables, for example, milk composition, milk temperature, somatic cell count, cow live weight, body condition score, lameness, etc. With a number of daily milkings at herd level of about 2.5, the AMS enables farmers to collect a huge amount of real time data on individual cows and their performances, in many cases at a quarter level, which allows for a detail in the monitoring never achieved so far.The big data generated by the multiple sensors implemented in the AMS can be statistically managed for the development of algorithms for the real-time detection of changes that can enable the farmer to implement timely and optimized management actions. AMS can be considered not only as an alternative to traditional milking systems but also as a new and general approach to manage dairy herd health and production efficiency . In partMastitis is the most important disease that affects dairy herds both in terms of prevalence and economic costs: it causes production losses, milk quality alterations, medication and extra labour costs, discarded milk, and early culling of animals . MoreoveIn this context, the aim of the study was to examine the variables automatically recorded by the AMS sensors, and some new derivative indicators, studying their association, changes over time, and ability to predict the risk of increasing milk somatic cell count.Four dairy cattle farms located in Lombardy , equipped with automatic milking systems of two different brands , were involved in the study. DeLaval’s Milking Robots is labelled “A AMS” and Lely’s Milking Robots is labelled “B AMS”. The dataset included information recorded by the AMSs in the period 2016-2019. A total of 602,000 records of 720 cows from the two farms using type A AMS, and 150,000 records of 283 cows from the two farms using type B AMS were collected. All the cows were kept in a free-stall housing, without access to pasture; lying cubicles were bedded with straw and sawdust. Cows were fed a partial mixed ration integrated by concentrate in the AMS stall. In From The two types of AMSs slightly differed for the data recorded and made available. In particular, B AMS registered a lot of variables on milking efficiency such as number of daily refusals, failures, and downtimes. Moreover, B AMS captured indicators of udder milk composition  and somatic cell count by sensors, based on NIR spectroscopy and viscosity, respectively. On the contrary, A AMS made available a lot of information per quarter such as milk yield and milk flow. Both systems measured milk electrical conductivity per quarter, but in the case of B AMS, conductivity was expressed as a score out of 100, instead of mS/cm. Consequently, variables studied were partly different for the two AMSs. 2 ((somatic cell count/mL)/12,500).In addition to the information obtained by the AMSs, milk production and quality data from individual monthly official milk controls performed by the National Breeders’ Association were included in the dataset. Milk samples were analysed for fat and protein contents  and milk somatic cell count (Fossomatic). Somatic cell count was log-transformed as Linear Score (LS), using the equation suggested by Wiggans and Shook : LS = loIndividual cow data as days in milk , stage of lactation  and number of lactation  were included in the dataset.Each individual cow record was classified as a function of somatic cell count obtained from official controls as LOW  or HIGH . Moreover, cows with more than 80% of the official milk analyses characterized by LSc ≤ 5  were classified as low SCC (LSCC); cows with more than 80% of the official milk analyses characterized by LSc > 5  were classified as high SCC (HSCC); in all the other cases cows were classified as fluctuating SCCc (FSCC).A number of new additional variables were calculated starting from the data recorded by the AMSs: for all the herds milk production rate at udder level, as rate of milk secretion per hour ; for the herds milked with A AMS, the within-cow within-milking percentage differences between individual quarters and the average of the four quarters of the minimum milk yield /meanYq * 100), the minimum average milk flow , the maximum milk electrical conductivity ; for the herds milked with B AMS, the within-cow within-milking percentage differences between the individual quarters and the average of the four quarters of the minimum milking times ; and the maximum milk conductivity score .The whole dataset was analysed using SAS software  always separately for the two types of AMS. Descriptive statistics and Pearson’s correlation analyses were obtained using Proc MEANS, Proc FREQ and Proc CORR. Differences between front and rear quarters and between right and left ones were evaluated using a GLM procedures.A GLM analysis was performed to test the differences among the three SCC cow classes in terms of ΔECq, ΔYq, ΔAMFq for the A AMS herds and in terms of ΔECSq and ΔTq for the B AMS herds. Number of lactation, stage of lactation, and farm effects were included in the models.Two types of multivariate analyses were performed: a Principal Component Analysis (PROC PRINCOMP), and a Logistic Analysis (PROC LOGISTIC).Through the Principal Component Analyses, the relationships among data from official controls, data recorded by the AMSs, and the additional variables calculated were studied for each AMS type.Class variables were created to perform the Logistic Analyses with the aim of identifying the main categorical variables associated with high level of milk SCC in the official controls . Dependent variables were MPR , ΔECq , ΔAMFq , ΔYq , ΔECSq , and ΔTq .In the Logistic Analyses, variables or combinations of variables (interaction terms) were excluded through a stepwise backward multiple regression method based on a 0.2 significance level. The results of the analyses built, for each AMS type, a final model including variables (risk factors) that were significantly associated with SCC > 300,000/mL. The final models were described in terms of odds ratios and 95% confidence intervals.The general descriptive statistics of the herds milked with the two AMS (A and B) are reported in The A AMS provides a lot of information on milk ejection at a quarter level. In particular average milk flow (1.13 ± 0.32 kg/min), peak milk flow (1.61 ± 0.38 kg/min), milk yield per milking (3.63 ± 1.22 kg/milking), and milk electrical conductivity (4.12 ± 0.35 mS/cm). According to Juozaitienė et al.  and KathThe B AMSs, in addition to some information at a quarter level, provide a lot of information on milking efficiency and indications about milk components and somatic cell count at udder level from the AMS sensors. The most important variables registered at quarter level were milking time (2.70 ± 1.17 min) and milk electrical conductivity expressed as score (69.2 ± 3.61). At udder level, average milk flow was 3.57 ± 1.10 kg/min and indications of whole udder milk components from sensors were 3.9 ± 0.83%, 3.4 ± 0.21%, and 5.00 ± 0.11% for milk fat, protein, and lactose, respectively. Milk somatic cell count from AMS sensors was 225,695 ± 451,540 cell/mL and the milk temperature was on average 39.1 ± 0.64 °C.For the herds milked with A AMS, the average % difference within-cow and within-milking of milk yield of the least productive individual quarters compared to the mean of the four quarters (ΔYq) was −24.2 ± 15.6%; the average % difference in average quarter milk flow (ΔAMFq) was −16.9 ± 10.7%; and the average % difference in quarter milk conductivity (ΔECq) was 3.73 ± 4.24%.For the herds milked with B AMS, the average % difference in milking time of individual quarters compared to the average of the four quarters (ΔMTq) was −25.1 ± 16.4%, and the average difference in electrical conductivity score (ΔECSq) was 2.22 ± 3.14%.In A AMS herds, rear quarters showed significantly higher milk yield in comparison to front quarters , higher average milk flows , and higher peak milk flows  in agreement with previous studies ,17,18. IIn comparison to the left quarters, right quarters produced significantly more milk  and had slightly higher average and peak milk flows in A AMS herds. In B AMS herds, right quarters showed significantly lower milking time . Differences in milk ejection among left and right quarters were noted also by Tancin et al. ; this coPearson’s correlation coefficients are shown in Lyons et al.  found a In the B AMS herds , similarMilk electrical conductivity score of each quarter (ECSq) was negatively correlated with lactose indication , but similar to what has been observed in A AMS herds, it did not show any correlation with LSc (r = 0.04 ÷ 0.07 for the different quarters) and SCCams . Number of daily milkings (nM) was positively correlated with MPR (r = 0.50) and negatively correlated with MI (r = −0.70). MPR showed a positive correlation with number of lactation  and a negative correlation with MI (r = −0.48), similar to what was seen in the A AMS and in agreement the findings of other authors ,22.Correlation coefficients between milk components from official controls and indications provided by the B AMS sensors were 0.45 and 0.41 for milk fat and milk protein percentages, respectively. Somatic cell count provided by the B AMS (SCCams) showed a positive correlation with LSc (r = 0.38). The r coefficient values for milk component and SCC show a medium degree of correlation between the results provided by the AMS and the lab. This may depend on a limit of the AMS sensors but also on the fact that the official control provides only monthly values that may not necessarily be representative of the trend.Although there was not any correlation among milk electrical conductivity score of single quarters (ECSq) and both LSc and SCCams, the % difference in milk electrical conductivity score between single quarters and udder (ΔECSq) was positively correlated to LSc (r = 0.27) and SCCams (r = 0.44).The results obtained from the PCA are plotted in The PCA on A AMS herds’ data a confirmThe analysis confirmed the positive association among LSc and % difference in milk conductivity (ΔECq) of single quarters vs. udder. These variables are in the opposite quadrant of the % difference in milk yield (ΔYq) and % difference in average milk flow (ΔAMFq) of single quarters vs. the whole udder.Similar results were obtained from the PCA on B AMS data b. Daily The indication of somatic cell count from AMS (SCCams) and linear score from official controls are very close on axis 1. Similar closeness emerges for milk fat and protein indications from AMS and milk fat and protein from official controls. These results suggest that the sensors for milk quality currently installed on many AMSs could be considered reliable enough for these parameters.The results from the PCA suggest that the distancing of a single quarter from the condition of the whole udder in terms of milk electrical conductivity (ΔECq or ΔECSq), milk yield (ΔYq), average milk flow (ΔAMFq), and milking time (ΔTq) is well related to SCC and LS and can be useful as indicators of udder health problems.In the A AMS herds, % difference milk electrical conductivity of quarter vs. the whole udder (ΔECq) was significantly influenced (P < 0.001) by the SCC cow class , nLACT, DIM, and SCCc. Percentage difference in quarter vs. udder milk yield (ΔYq) was significantly affected (P < 0.001) by the SCC cow class , nLACT, DIM, and herd. Similarly, in the B AMS herds, % difference in quarter vs. udder milk conductivity score (ΔMCSq) was significantly influenced (P < 0.001) by the SCC cow class, nLACT, DIM, herd, and SCCc.This is in agreement with the results of the PCA where increasing ΔECq, where associated to higher LS al the official control. This result suggests that the increase in difference between the milk EC of a single quarter and the mean values of the four quarters in the days before the official milk control could be a good indicator of the risk to have a result of high SCC and can be useful as an early predictor of altered mammary health.In A AMS herds , % diffeInzaghi et al.  noted thLogistic analyses were performed to identify the main risk factors for having a somatic cell count at the official control (SCCc) higher than 300,000 cells/mL. The logistic analysis showed also that when milk yield of the single quarter sensibly diverges from the mean value of the four quarters (ΔYq ≤ −24%) the risk of having high SCCc  at the milk control increases by 1.18 times in comparison with the case of ΔYq > −24%. Lastly, a lower risk (0.80) of high SCCc is associated with % difference in quarter vs. udder average milk flow (ΔAMFq) higher than −15%.3 cells/mL) had lower peak flow rate compared with quarters with low SCC (<200 × 103 cells/mL). For AMS herds, Hammer et al. [The relationship between the risk of mastitis or high somatic cells and average or peak milk flow is controversial. Grindal et al.  suggester et al.  reportedr et al. , a widerIn the B AMS herds , besidesThe big data generated by the multiple sensors implemented in the automatic milking systems can be statistically managed to develop algorithms for the real-time detection of changes that may be indicative of mastitis or impaired health and welfare conditions of dairy cows. The results of the study showed that some variables automatically recorded by the AMSs at a quarter level can be used as good indicators of high SCC risk, when the deviation among the single quarters and the average of the four quarters is considered. Looking at the temporal patterns, the quarter vs. udder percentage difference in milk electrical conductivity showed an increase in the fourteen days preceding an official milk control higher than 300,000 cells/mL. Similarly, deviations over time in quarter vs. udder milk yield, average milk flow, and milking time emerged as potential indicators for high somatic cell count. The Logistic Analysis showed that Milk Production Rate (kg/h) and the within-cow within-milking percentage variations of single quarter vs. udder milk electrical conductivity, milk yield, and average milk flow are all risk factors for high milk somatic cell count.The result suggests that these variables, alone or in combination, and their progression over time, could be used to improve the early prediction of risk situations for udder health in AMS milked herds.