Ecology

AbstractThis study investigated the interactive effects of salinity levels (0‰, 18‰, and 36‰) and stocking densities (50, 100, 150, and 200 fish/m3) on water quality, growth performance, physiological responses, and economic returns of red tilapia (Oreochromis spp., initial weight of 12.33 ± 2.51 g/fish) reared in a biofloc technology (BFT) system using saline groundwater. A 3 × 4 factorial design with 36 fiberglass tanks (1 m3 each) was employed for 6 months. Key water quality indicators, fish growth indices, hematological and biochemical markers, antioxidant enzymes, immune parameters, and economic performance metrics were assessed. Results showed that increasing salinity and density significantly reduced dissolved oxygen (DO) levels and increased total ammonia nitrogen (TAN), NH3, NO2, and NO3 concentrations (p < 0.001). Biofloc volume (BFV) increased with stocking density across salinities, peaking at 44.4 ± 1.06 mL/L at 0‰ and 200 fish/m3, while higher salinity (36‰) generally reduced BFV. Variations in biofloc composition (protein 22–33%) and fish muscle composition (protein and lipid reduction at 36‰ and 200 fish/m3) indicated metabolic adjustments under stress. The highest final weight (261 ± 1.69 g/fish) was observed at 36‰ salinity with low stocking density (50 fish/m3), whereas the most favorable combination of growth rate, feed conversion ratio, and protein efficiency ratio occurred at 18‰ salinity and moderate stocking densities (100–150 fish/m3). Growth performance and feed utilization declined markedly at 36‰ with high density (200 fish/m3). Hematological indicators (RBC, Hb, Hct) and immune biomarkers (lysozyme, IgM, complement C3) were suppressed at extreme salinity-density combinations, while oxidative stress (high MDA) and hepatic dysfunction (elevated AST and ALT) were evident. Economic analysis confirmed that 18‰ salinity with 200 fish/m3 yielded the highest profit (1000 ± 54.8 EGP/treatment) and lowest operating ratio, while 150 fish/m3 at the same salinity provided slightly lower profit but better fish welfare indicators and immune responses, whereas high-density and hypersaline conditions reduced profitability due to poor growth and increased feed costs. In conclusion, 18‰ salinity combined with 100–150 fish/m3 provides the optimal balance between biological performance, fish welfare, and economic viability in red tilapia BFT systems. These findings offer evidence-based guidelines for sustainable inland saline aquaculture, supporting enhanced production efficiency and profitability in arid and saline-prone regions.

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Download referencesFundingOpen access funding is provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).Author informationAuthors and AffiliationsNational Institute of Oceanography and Fisheries (NIOF), Cairo City, EgyptGhada R. SallamDepartment of Animal and Fish Production, Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria City, 21531, EgyptMohamed Hamdy, Samy Y. El-Zaeem, Walied M. Fayed & Akram Ismael ShehataDepartment of Animal Production, Faculty of Agriculture, Tanta University, Tanta City, 31527, EgyptMohammed F. El BasuiniFaculty of Desert Agriculture, King Salman International University, Sinai City, South Sinai, EgyptMohammed F. El BasuiniDepartment of Medical Analysis, Faculty of Applied Science, Tishk International University, Erbil City, IraqYusuf Jibril HabibDepartment of Animal and Poultry Production, Faculty of Agriculture, Damanhour University, Damanhour City, 22516, EgyptEslam TefalAuthorsGhada R. SallamView author publicationsSearch author on:PubMed Google ScholarMohamed HamdyView author publicationsSearch author on:PubMed Google ScholarMohammed F. El BasuiniView author publicationsSearch author on:PubMed Google ScholarSamy Y. El-ZaeemView author publicationsSearch author on:PubMed Google ScholarYusuf Jibril HabibView author publicationsSearch author on:PubMed Google ScholarWalied M. FayedView author publicationsSearch author on:PubMed Google ScholarEslam TefalView author publicationsSearch author on:PubMed Google ScholarAkram Ismael ShehataView author publicationsSearch author on:PubMed Google ScholarContributionsGhada R. Sallam: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data Curation, Visualization, Writing – Review & Editing. Mohamed Hamdy: Methodology, Validation, Formal analysis, Investigation, Resources, Data Curation. Mohammed F. El Basuini: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data Curation, Visualization, Supervision, Writing – Original Draft, Writing – Review & Editing. Samy Y. El-Zaeem: Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data Curation, Visualization. Yusuf Jibril Habib: Formal analysis, Data Curation, Writing – Review & Editing. Walied M. Fayed: Methodology, Formal analysis, Investigation, Visualization. Eslam Tefal: Methodology, Software, Validation, Formal analysis, Data Curation, Original Draft, Writing – Review & Editing. Akram Ismael Shehata: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data Curation, Visualization, Supervision, Writing – Original Draft, Writing – Review & Editing.Corresponding authorsCorrespondence to
Mohammed F. El Basuini or Akram Ismael Shehata.Ethics declarations

Competing interests
The authors declare no competing interests.

Ethical approval
All experimental procedures were reviewed and approved by the Animal Use Ethics Committee of Alexandria University (protocol number AU:19/24/06/11/1/34). The study was conducted following the ARRIVE guidelines v2.0, ensuring compliance with internationally accepted ethical standards for the care and use of animals in research. Fish were handled carefully to minimize stress during all experimental procedures, and no unnecessary harm was inflicted.

Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Reprints and permissionsAbout this articleCite this articleSallam, G.R., Hamdy, M., El Basuini, M.F. et al. Optimizing salinity and stocking density for red tilapia in zero-water-exchange biofloc system: integrated performance, physiological, and economic assessment.
Sci Rep (2025). https://doi.org/10.1038/s41598-025-28812-xDownload citationReceived: 12 August 2025Accepted: 12 November 2025Published: 18 December 2025DOI: https://doi.org/10.1038/s41598-025-28812-xShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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KeywordsBiofloc technology (BFT)Red tilapia (Oreochromis spp.)SalinityStocking densityGrowth performanceHematological biomarkersAntioxidant enzymesImmune response More

AbstractSundarbans region is an assemblage several delta formed in Bay of Bengal with largest concentration of mangrove which plays a crucial role in mitigating the impact of climate change with large ecosystem. The mangrove ecosystem demands further investigations to assess the vulnerability of vegetation. In context of present environmental change, the existing vegetation of Sundarbans is threatened by natural and human induced factors. This study incorporated these issues by analysing the vulnerability of mangrove forest in Indian Sundarbans deltaic region. To assess the vegetation condition, various vegetation indices are used including Normalised Difference Vegetation Index (NDVI), Transformed Normalised Difference Vegetation Index (TNDVI), Green Chlorophyll Index (GCI), Chlorophyll Vegetation Index (CVI), Soil Adjusted Vegetation Index (SAVI), and Atmospherically Resistant Vegetation Index (ARVI) etc. These indices are calculated using remote sensing satellite data of 2010 and 2020. Vulnerability has been assessed through vegetation health assessment by spatial modelling with the data from aforesaid vegetation indices. The result shows that specific regions have experienced an increase in stressed vegetation condition accompanied by the problems such as waterlogging and expanding areas under aquaculture. Furthermore the area under healthy vegetation has significantly decreased between 2010 and 2020.

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Data availability

Remote Sensing Satellite data has been used in this study for the year 2010 and 2020. The data for different bands (Blue, Green, Red and NIR) of Landsat 5 TM and Landsat 8 OLI have been collected from USGS (https://earthexplorer.usgs.gov/) for the month of December for both the years with 30 m spatial resolution (Table 1). The satellite imageries of different bands have been analyzed with QGIS Software. The data further has been modified by different vegetation indices and vegetation health assessment with spatial modeling. For the validation of the results from the analysis of satellite imageries, this study has also incorporated Google Earth historical images of specific locations along with some field visits.
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Amlan Ghosh or Padmaja Mondal.Ethics declarations

Competing interests
The authors declare no competing interests.

Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Reprints and permissionsAbout this articleCite this articleGhosh, A., Mondal, P. Analysing the vulnerability of mangrove forest by vegetation health assessment: a study of Indian sundarbans deltaic region.
Sci Rep (2025). https://doi.org/10.1038/s41598-025-26905-1Download citationReceived: 13 June 2025Accepted: 30 October 2025Published: 18 December 2025DOI: https://doi.org/10.1038/s41598-025-26905-1Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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KeywordsMangrove vulnerabilityVegetation indicesVegetation health condition More

AbstractMonitoring microclimatic conditions in underground environments is crucial for understanding chemical and biological processes occurring in caves and their effect on archaeological, palaeontological, and palaeobotanical records. The Israel Cave Climate Project (ICCP) dataset provides high-resolution microclimatic data from twelve caves across three climate zones — Desert, Steppe, and Mediterranean — measured during 2019–2021 using a uniform protocol. All twelve are natural karstic caves containing diverse, rich, and typically multi-period archaeological records. Within each cave, hourly air temperature and relative humidity measurements were recorded over a year, and these data are presented here in full. The physical and speleological characteristics of the studied caves and the content and nature of their archaeological records are also detailed. The combined high-resolution datasets, incorporating speleological, climatological, and archaeological records, provide unparalleled raw data valuable for studying cave environments, particularly cave archaeology, site formation processes, and preservation and conservation of ancient material and bioarchaeological records.

Data availability

The dataset is available at Zenodo (https://doi.org/10.5281/ZENODO.17505739)51.
Code availability

While the netCDF data file51 can be operated and managed in any suitable software, we also offer the Shiny-based ICCP package built for R v. 4.5.2, that we wrote to overview the data file along with the comparative CHELSA data. The code is open-source and can be found either on Zenodo51, or GitHub repository.
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Micka Ullman or Mitya Kletzerman.Ethics declarations

Competing interests
The authors declare no competing interests.

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Reprints and permissionsAbout this articleCite this articleUllman, M., Kletzerman, M., Oron, A. et al. Year-round hourly temperature and humidity sensor readings from arid caves, Judean Desert, Israel.
Sci Data (2025). https://doi.org/10.1038/s41597-025-06420-8Download citationReceived: 09 June 2025Accepted: 03 December 2025Published: 17 December 2025DOI: https://doi.org/10.1038/s41597-025-06420-8Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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AbstractHigh-resolution data of individual trees are critical for advancing forest monitoring, inventory development, and ecological research. This dataset, BioDiv-3DTrees, comprises 4,952 individual tree point clouds of 19 species, captured using Terrestrial Laser Scanning (TLS) and Unmanned Aerial Vehicle Laser Scanning (ULS), along with 3,386 Quantitative Structure Models (QSMs) and graph representations of the 14 broadleafed species in the dataset. The trees were sampled across the three research areas of the Biodiversity Exploratories in Germany. Each tree is linked to an existing open-access forest inventory dataset, which includes species identity, diameter at breast height (DBH), and tree height. The dataset is suitable for various research applications, including biomass estimation, algorithm development, tree structure analysis, and data fusion with traditional inventory methods. All QSMs were generated using TreeQSM 2.4.1 and have been validated for tree height, diameter at breast height and crown projection area against their underlying point clouds to ensure consistency. The dataset provides a reliable and scalable resource for forest science and remote sensing communities.

Data availability

The complete dataset is available on GROdata (https://doi.org/10.25625/8PB1IF).
Code availability

The code of the used histogram-based outlier removal algorithm, as well as the code to reverse the coordinate normalization is available at the dataset’s GitLab repository (https://gitlab.gwdg.de/griese1/biodiv-3dtrees/). The repository also includes the code that creates and cleans up the graph representation.
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Download referencesAcknowledgementsWe thank Tim Geis, Muluken Bazezew, Tao Jiang and Hans Fuchs for their help during the data acquisition and point cloud post processing. We thank the authors of the forest inventory dataset Peter Schall, Christian Ammer, as well the data collector Andreas Parth for their work, which made it possible to add tree species labels to this dataset. We thank the managers of the three Exploratories, Max Müller, Robert Künast, Franca Marian, and all former managers for their work in maintaining the plot and project infrastructure; Victoria Grießmeier for giving support through the central office, Andreas Ostrowski for managing the central data base, and Markus Fischer, Eduard Linsenmair, Dominik Hessenmöller, Daniel Prati, Ingo Schöning, François Buscot, Ernst-Detlef Schulze, Wolfgang W. Weisser, and the late Elisabeth Kalko for their role in setting up the Biodiversity Exploratories project. We thank the administration of the Hainich national park, the UNESCO Biosphere Reserve Swabian Alb, and the UNESCO Biosphere Reserve Schorfheide-Chorin as well as all land owners for the excellent collaboration. Field work permits were issued by the responsible state environmental offices of Baden-Württemberg, Thüringen, and Brandenburg. The work has been partly funded by the DFG Priority Program 1374 “Biodiversity-Exploratories” (DFG project numbers 433273584 and 193957772). Funding for Nils Griese was provided by the German Research Foundation (DFG project number 496533645). This project has received funding from the European Research Council (ERC) under the European Union’s Horizon Europe research and innovation program (Grant agreement No. 101041669).FundingOpen Access funding enabled and organized by Projekt DEAL.Author informationAuthors and AffiliationsDepartment of Forest Inventory and Remote Sensing, University of Göttingen, Göttingen, GermanyNils Griese & Nils NölkeInstitute of Computer Science and Campus Institute Data Science, University of Göttingen, Göttingen, GermanyMartin RitzertAuthorsNils GrieseView author publicationsSearch author on:PubMed Google ScholarMartin RitzertView author publicationsSearch author on:PubMed Google ScholarNils NölkeView author publicationsSearch author on:PubMed Google ScholarContributionsN.G. collected the data. N.G. developed the methods described apart from the Graph derivation. M.R. derived the Graphs from the data and contributed to the data validation. N.N. supervised the project. All authors discussed the results and contributed to the final manuscript.Corresponding authorCorrespondence to
Nils Griese.Ethics declarations

Competing interests
The authors declare no competing interests.

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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Reprints and permissionsAbout this articleCite this articleGriese, N., Ritzert, M. & Nölke, N. A large dataset of labelled single tree point clouds, QSMs and tree graphs.
Sci Data (2025). https://doi.org/10.1038/s41597-025-06421-7Download citationReceived: 04 July 2025Accepted: 03 December 2025Published: 17 December 2025DOI: https://doi.org/10.1038/s41597-025-06421-7Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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AbstractUrban rewilding is increasingly recognized as a nature-based solution for restoring biodiversity, mitigating climate risks, and strengthening urban resilience. Yet, empirical evidence on how rewilding is perceived and supported by both policymakers and the public—particularly in post-socialist contexts—remains scarce. This study investigates expert and community perspectives on urban rewilding in Poland through a mixed-method design: a nationwide survey of 32 municipal environmental officials and a visual preference survey with 1,000 residents of the coastal city of Sopot. Expert responses highlight strong conceptual support for rewilding’s ecological and social benefits, but also identify persistent concerns about institutional feasibility, funding, and integration into existing planning frameworks. Community results reveal consistent public endorsement of moderate rewilding, with more cautious acceptance of intensive ecological designs in highly symbolic civic spaces. Taken together, the findings suggest that urban rewilding in Central and Eastern Europe is both socially viable and ecologically desirable, but its successful implementation will depend on adaptive governance, participatory planning, and the strategic use of visual engagement tools to bridge policy ambition with public expectations.

Data availability

The data that support the findings of this study are openly available in the Figshare repository at [https://doi.org/10.6084/m9.figshare.27089560](https:/doi.org/10.6084/m9.figshare.27089560) .
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Reprints and permissionsAbout this articleCite this articleCirella, G.T., Kempa, J., Paczoski, A. et al. Stakeholder perceptions and planning implications for urban rewilding as a nature-based solution in Poland.
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KeywordsCircular land useCommunity perceptionsEnvironmental governancePublic spaceRewilding policySustainable urban planning More

AbstractThis study characterized a green-waste-derived biochar from date palms and ghaf trees and investigated its potential as a soil amendment with halotolerant Bacillus spp. to improve tomato seedling quality under dual stress of salinity and nutrient deficiency. Biochar was produced through pyrolysis at 450 °C and then characterized for yield, pH, electrical conductivity, proximate analysis, surface morphology, energy-dispersive X-ray spectroscopy, and heavy-metal content. Its effectiveness was tested both alone and in combination with a Bacillus sp. mix, using a completely randomized design with varying NPK fertilizer levels and saline irrigation. Tomato seedlings were evaluated 45 days after planting for various vegetative, morphological, physiological, and nutrient content indicators. Under normal conditions, applying biochar combined with a Bacillus mix at 0% NPK greatly enhanced all measured parameters, often exceeding values observed with 100% NPK fertilization. This approach was especially effective under saline irrigation, resulting in significant increases in morphological parameters (40–150%), physiological parameters (51–94%), and nutrient content (34–63%) compared to control plants that received 100% NPK. Additionally, this treatment resulted in a 42% decrease in sodium accumulation. Using the biochar with the Bacillus mix effectively replaces chemical fertilizers and enhances salinity tolerance, supporting sustainable farming through waste recycling and less dependence on fertilizers.

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The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
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KeywordsTomato seedlingsSustainabilityChemical fertilization
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Abstract

The milk thistle plant strengthens the body’s immune system and is one of the top-selling medicinal plants in the world in terms of turnover. Considering the water crisis in Iran and especially in the northwest of Iran, which has led to the drying up of Lake Urmia, the cultivation of crops with low water requirements is considered important under the conditions of water scarcity. The cultivation of medicinal plants will solve the environmental problems of the region, change the cropping pattern from high-consumption to low-consumption crops, increase farmers’ income and ultimately improve the living conditions of farmers in the region. Given the significance of milk thistle, an experiment was conducted to assess the effect of irrigation type and biofertilizers on its seed yield, oil yield, and fatty acids. So, a split-plot experiment was conducted in Miandoab County at the south of West Azerbaijan province, Iran in 2016–2017 based on a randomized complete block design with 10 treatments and three replications. The main plot was assigned to irrigation regime (at two levels of full irrigation and no irrigation) and the sub-plot to biofertilizer (at five levels of control, nitrogen biofertilizer, phosphorus biofertilizer, potash biofertilizer, and NPK biofertilizer). Based on the results, the interaction of irrigation × biofertilizer was significant for biological yield, capitule number, and DPPH radical scavenging at the P < 0.01 probability level and for seed yield per plant and plant height at the P < 0.05 probability level. But, this interaction was insignificant for the other traits including harvest index, crown diameter, branch number, oil yield, phenol and flavonoid content, superoxide radical scavenging, nitric oxide radical scavenging, and chain breaking. In addition, it was found that most morphological and biochemical traits of the thistles were affected by irrigation and biofertilizers so that the biofertilizers improved them versus the control. Given the significant role of biofertilizers in the development of sustainable agriculture, they can be a good candidate in case there is a need for nutrient supplements for this plant. Under drought stress conditions, the combination of bio-fertilizers (NPK(b)) and irrigation helps the plant to tolerate drought stress easily and also improves the medicinal properties of the plant.

IntroductionMilk thistle (Silybum marianum L.) is an annual herbaceous plant species originated from the east of the Mediterranean region1, but it is extensively distributed in hot and arid regions. Today, this plant species has provoked interests due to its significance in pharmaceutical industries (treatment of liver diseases) and plant oil production2,3. It is used in the treatment of hepatic disorders, hepatitis (A), high blood cholesterol content, some toxicities, bilious precipitations and stones, migraine, spleen diseases, and alcoholic cirrhosis and has antiviral, anti-coagulation and anti-thrombotic activities4.The medicinal plants the black cumin (Nigella sativa L.), the echinacea purpureae (L.) moench and the milk thistle (Silybum marianum L.) are effective in strengthening the body’s immunity and in treating hepatitis diseases. It should be noted that black cumin is not very well known in the world and has a low turnover, and the water consumption of echinacea is also higher5,6. Due to the reasons mentioned, the milk thistle was selected for the present study. Oil, food, cosmetics, and forage uses are some other applications of thistle. Drought stress is one of the most important and destructive abiotic stresses influencing plant production. It is estimated that about 26% of all arable lands of the world are located in arid regions7. On the other hand, we have witnessed the loss of crop quality and soil fertility in the recent decades due to the inappropriate and excessive use of fertilizers and chemical pesticides and the crisis of the environment pollution, especially the pollution of water and soil resources, which entails the contamination of food and endangers the safety of human communities. Furthermore, the inadequate supply of nutrients8 and irrigation water9 dramatically disturb plant growth and physiology. Hence, reductions in yields and quality associated the unsuitable water and fertilization management10. So, the focus has dramatically been shifted towards finding approaches to improving soil and crop quality and removing pollutants. The prevention of the excessive application of chemical inputs and sustainable food production along with environmental conservation are issues that have drawn the attention of most researchers and crop producers11,12. In this respect, biofertilizer-based sustainable farming has been considered more than ever to stop or reduce the consumption of chemical inputs13,14.In a study on the combined effect of drought stress and biofertilizer inoculation on the quantitative and qualitative traits of thistle, Mohammadpour Vashvaei et al.15 reported that the simple and interactive effects of drought stress and biofertilizers were significant for all traits. It has also been reported that the use of biofertilizers induced plant growth by increasing nutrient availability and thereby it increased yield components (capitule number per plant, seed number per capitule, and 1000-seed weight), which resulted in the enhancement of thistle yield16. Volaii et al.17 found the positive impact of biofertilizer and vermicompost on yield and yield components of thistle and stated that the concurrent use of the two biofertilizers was most effective.In another study, Eskandari Nasrabadi et al.13 revealed promising results as to the reduction of chemical fertilizer use in the cultivation of thistle and reported the need to consider more use of biological and organic fertilizers. Based on the results of a four-year experiment by Haban et al.18 about the negative impact of chemical fertilizers on thistle seed yield in the long run, it was recommended to use biofertilizers. However, Yazdani Biuki et al.19 reported that none of the morphological traits and yield components of thistle was influenced by different fertilizer treatments. They hypothesized that thistle may not easily lend itself to the effect of fertilizers.The milk thistle plant strengthens the body’s immune system and is one of the top-selling medicinal plants in the world in terms of turnover. Considering the water crisis in Iran and especially in the northwest of Iran, which has led to the drying up of Lake Urmia, the cultivation of crops with low water requirements deficit irrigation tactic is considered important under the conditions of water scarcity20. However, irrigating plants with water less than normal level undoutly results in decline in crop productivity and quality21. Therefore, under these circumstances, unconventional strategies should be adopted. The cultivation of such crops will solve the environmental problems of the region, change the cropping pattern from high-consumption to low-consumption crops, increase farmers’ income and ultimately improve the living conditions of farmers in the region. If this scientific research is not carried out, the northwestern region of Iran will be deserted and devoid of inhabitants in the next 40 years due to salt storms in Lake Urmia. This is because toxic salt particles are dispersed in the atmosphere and are the most important factor in the occurrence of respiratory diseases, various types of cancer and skin and digestive problems. These particles also have an impact on eye health, heart health and high blood pressure. Since milk thistle is of high importance due to its drought stress adaptation and resistance and nowadays the use of biofertilizer to curb on the use of chemical fertilizer and to increase crop yields is important for the movement towards sustainable agriculture, which can be much more important under no-irrigation conditions22 and since little research has addressed the effect of combined use of no-irrigation conditions and biofertilizers on medicinal plants23 including milk thistle, the present study focused on the effect of bio-fertilizers on the quality and quantity of thistle under no-irrigation conditions. Bio-fertilizers effectively increase the plant’s tolerance to drought stress, prevent a reduction in grain yield and improve the plant’s antioxidant properties (phenolic and flavonoid compounds).Results and discussionThe results of ANOVA are presented in Table 1 for morphological traits and in Table 2 for biochemical traits. Accordingly, the interaction of irrigation × biofertilizer was significant for biological yield, capitule number, and DPPH radical scavenging at the P < 0.01 level and for seed yield per plant and plant height at the P < 0.05 level. But, it was not significant for the other traits including harvest index, crown diameter, branch number, oil yield, phenol and flavonoid content, superoxide radical scavenging, nitric oxide radical scavenging, and chain breaking.Table 1 The results of the analysis of variance for the recorded morphological traits based on a split-plot experiment.Full size tableTable 2 The results of the analysis of variance for the recorded biochemical traits based on a split-plot experiment.Full size tableSeed and biological yield per plantBased on the comparison of the means, the treatment of the P biofertilizer to the fully-irrigated plants was related to the highest seed yield (20.74 g), and the non-irrigated plants that were not treated with a biofertilizer produced the lowest one (10.99 g). The interaction of ‘P biofertilizer × full irrigation’, which produced the highest seed yield per plant, did not differ significantly only from the treatments of the NPK biofertilizer and the N biofertilizer to the full irrigation conditions. Among the fully-irrigated plants, those treated with the P biofertilizer exhibited the highest seed yield (20.74 g) and those not treated with a biofertilizer exhibited the lowest one (17.68 g) so that only P biofertilizer was grouped with the control in a separate statistical group, and the other treatments did not show a significant difference from the control. Under the no-irrigation conditions, the highest and lowest seed yields were 16.78 and 10.99 g obtained from the NPK biofertilizer and no-biofertilizer, respectively. In general, the bio-fertilizer treatment with full irrigation and without irrigation increased grain yield by about 15 and 35%, respectively, compared to the control treatment. In these conditions, in addition to the NPK biofertilizer, the P biofertilizer differed from the control significantly too whereas the N biofertilizer and the K biofertilizer were in the same statistical group with the control. So, it can be concluded that the NPK fertilizer could partially alleviate the adverse impacts of drought stress. In other words, although when less water is consumed, which naturally induces drought stress and seed yield per plant is decreased, the application of the NPK biofertilizer can partially mitigate the negative impacts on the yield. This may partially be attributed to the positive effect of biofertilizers on improving plant nutritional status under stressful conditions24,25. It seems that the separate use of the N and K biofertilizers entailed responses similar to the control.In a similar finding, Mohammadpour Vashvaei et al.15 attributed the decline in seed yield of milk thistle under water deficit conditions to the reduction of photosynthesis and assimilation in the plants and the decline in the nutrients mobilized from the leaves to the seeds. Volaii et al.17 reported the positive effect of biofertilizer and vermicompost on yield and yield components of milk thistle and stated that the combined use of the two biofertilizers had the strongest impact on the traits. In another study, Eskandari Nasrabadi et al.13 found promising results as to the decrease in chemical fertilizer use in the cultivation of milk thistle. They emphasized that more attention should be given to the use of biofertilizers and organic fertilizers.The interaction of ‘NPK biofertilizer × full irrigation’ had the highest and ‘no biofertilizer × no irrigation’ had the lowest biological yield per plant (132 and 86.2 g plant−1, respectively). Under the full irrigation conditions, in addition to the NPK biofertilizer, the N biofertilizer produced a considerably high biological yield of 127 g plant−1. As expected, the lowest biological yield (116 g plant−1) was obtained from the full irrigation of the control plants, which was in the same statistical group with the P and K biofertilizers. Under no irrigation conditions, the NPK biofertilizer had the highest biological yield of 118 g plant−1 followed by the N biofertilizer, which produced 102 g plant−1 of biological yield. The other treatments did not differ from the control significantly (Fig. 1). In general, the bio-fertilizer treatment with full irrigation and without irrigation increased biological yield by about 11.5 and 27%, respectively, compared to the control treatment. It has been documented that low water supply reduced the availability of nutrients and uptake26, while reactive oxygen species are stimulated which damage the cell membrane with reduction of photosynthesis efficiency27.Sanchez et al.28 reported for Plantago major L. and P. lanceolota L. that the application of biofertilizers enhanced biological yield. Rodriguez et al.29 attributed the decline in biological yield of the stressed plants to the dry weight of their roots, stems, and leaves. Consistently, Anwar et al.30 observed that the application of NPK fertilizer + vermicompost increased the biological yield of basil versus the control. Yazdani Biuki et al.19 reported that the changes in the biological yield and harvest index of milk thistle in response to chemical, biological, and organic fertilizers versus the control were statistically significant.Fig. 1The results of means comparison for the interaction of irrigation × biofertilizer for biological yield per plant. Note: N(b): nitrogen bio-fertilizer, P(b): phosphate bio-fertilizer, K(b): potash bio-fertilizer, NPK(b): complete bio-fertilizers.Full size imageHarvest indexIt was found that the P biofertilizer had the highest harvest index of 16.3% and the N biofertilizer had the lowest one of 13.8%. The other treatments were placed in the same statistical group with the N biofertilizer despite some small variations (Fig. 2). This is consistent with the results of Darzi et al.31 for fennel and Rezaei-Chiyaneh et al.32 for Lallemantia as to the increase in yield and yield components of these plant species in response to the treatment of the biological phosphate fertilizer.Fig. 2The results of means comparison for the simple effect of biofertilizer on harvest index. Note: N(b): nitrogen bio-fertilizer, P(b): phosphate bio-fertilizer, K(b): potash bio-fertilizer, NPK(b): complete bio-fertilizers.Full size imagePlant heightThe treatment of N biofertilizer to the fully-irrigated plants was associated with the highest plant height of 178 cm and the control treatment under no-irrigation conditions was associated with the lowest plant height of 111 cm. Under the full irrigation conditions, the NPK and P biofertilizers were grouped in the same statistical category with the N biofertilizer, whereas the treatment of no biofertilizer had the lowest plant height of 153 cm and was significantly different from the other treatments. Under no irrigation conditions, the NPK biofertilizer had the highest plant height (138 cm) and the N biofertilizer had the second highest plant height, but the other treatments did not differ from the control significantly (Fig. 3a). In general, the bio-fertilizer treatment with full irrigation and without irrigation increased plant height by about 14 and 20%, respectively, compared to the control treatment. The morphological changes in medicinal plants in response to drought stress have been subject to extensive studies. Our findings are in agreement with the findings as to the decline in the plant height of basil33 and savory34 under drought stress. The significant impact of fertilizer treatments on plant height of milk thistle has already been reported35.Crown diameterThe mean crown diameter was higher in the full irrigation conditions (23.3 mm) than in the no-irrigation conditions (19.3 mm). The NPK biofertilizer exhibited the highest crown diameter of 22.6 mm followed by the N biofertilizer in the second rank (22.4 mm). These two treatments were significantly different from the control (19.3 mm) whereas this difference was not observed in the P and K biofertilizers (Fig. 3b). The quantity and quality of a plant’s vegetative growth depend on cell division, elongation, and differentiation, and all these are influenced by drought stress36. For the same reason, the crown diameter was lower in the no-irrigation conditions than in the full-irrigation conditions. Since the crown is a sink of assimilates, the improvement of nutritional conditions by the studied fertilizers increased assimilates and this increased stem diameter.Capitule numberBased on the results of the comparison of means, when drought stress was applied, almost five capitules were decreased from all plants. All treatments, including the control and biofertilizers, produced their highest capitule number (15 capitules) in the full irrigation conditions and they were all categorized in the same statistical group. However, the differences between the fertilizer treatments with one another and with the control were more considerable in the no-irrigation conditions so that the NPK biofertilizer was related to the highest number of capitules (13.3 capitules) and the control to the lowest number (8.7 capitules). Also, the N and P biofertilizers were placed in the same group and had an intermediate number of capitules. Only, the K biofertilizer did not differ from the control significantly (Fig. 3c). In general, the bio-fertilizer treatment with full irrigation and without irrigation increased capitule number by about 2 and 35%, respectively, compared to the control treatment. Irrigation is effective in increasing the number of capitules per plant due to its effect on increasing moisture and water uptake by the plants and improving nutrient uptake and retention capacity. On the other hand, drought stress limited plant growth and reduced the number of auxiliary branches, thereby reducing the number of capitules per plant.Volaii et al.17 concluded that organic and biological fertilizers were influential on most growth traits of milk thistle including the number of capitules per plant and capitule diameter. Mohammadpour Vashvaei et al.15 mentioned the number of capitule per plant to be the most important seed component. They revealed that this trait was significantly (P < 0.01) influenced by drought stress, biofertilizer, and their interaction. Most researchers agree that this trait is genetic and is influenced by genotype37.Fig. 3The results of means comparison for the interaction of irrigation × biofertilizer for plant height (a), crown diameter (b) and capitule number (c). Note: N(b): nitrogen bio-fertilizer, P(b): phosphate bio-fertilizer, K(b): potash bio-fertilizer, NPK(b): complete bio-fertilizers.Full size imageNumber of auxiliary branchesThe plants fully irrigated produced more branches (3 auxiliary branches) than those not irrigated (2.69 auxiliary branches). On the other hand, the application of the NPK and N biofertilizers was related to the highest number of auxiliary branches (3.11 and 3 auxiliary branches, respectively) and the control had the lowest number (2.46 auxiliary branches). But, the P and K biofertilizers were in between these two extremes (Fig. 4a). The increase in the number of auxiliary branches in the full irrigation conditions is related to the improved vegetative growth of the plant. The increased number of auxiliary branches in the fertilizer treatments can be caused by the improved growth conditions due to the uptake of nutrients, especially N. A study on the effect of biofertilizers (nitrogen and phosphate solubilizing microorganisms) on the quantitative and qualitative yield of milk thistle revealed that although biofertilizers enhanced some recorded traits, there were no significant differences among the fertilizer treatments in the number of branches per plant, capitule diameter, the number of capitule per plant, and 1000-seed weight17.Oil yield per plantAccording to Fig. 4b, oil yield was higher under the full irrigation conditions (5.2 g plant−1) than the no-irrigation conditions (3.5 g plant−1). Among the biofertilizer treatments, the NPK biofertilizer (4.94 g plant−1) and the P biofertilizer (4.83 g plant−1) had the maximum and the no-biofertilizer treatment had the minimum oil yield (3.68 g plant−1). A study reported that the soil application of various organic and chemical fertilizers was not influential on yield components and morphological traits of milk thistle, but they influenced oil percentage and seed silymarin and silybin contents so that the plants treated with compost, Azotobacter, and their mixture produced the highest seed oil percentage. In addition, the plants treated with chemical fertilizer had the lowest silybin content19. Finally, the acceptable oil percentage of milk thistle implies its high medicinal and nutritional significance. Based on our results, it can be considered a source of plant oil production.Fig. 4The results of means comparison for the simple effect of irrigation and biofertilizer on the number of auxiliary branches (a) and oil yield per plant (b)..Note: N(b): nitrogen bio-fertilizer, P(b): phosphate bio-fertilizer, K(b): potash bio-fertilizer, NPK(b): complete bio-fertilizers.Full size imageTotal phenol and flavonoid contentIn the present study, the null hypothesis as to the equal total phenol content of the different treatments was supported. In contrast, some research has shown than the accumulation of phenol compounds is very sensitive to nutrient stress so that total phenol content usually increases with the decrease in N content of the medium, but higher amounts of N in the medium usually stimulate plant growth and hinder phenol synthesis38. Mazarie et al.39 found that as drought stress was intensified, the phenol content of common sage was increased. The mean phenol content of milk thistle in the present study was 30.3 mg quercetin per g dry matter.Based on the observations and the comparison of means, flavonoid content was the maximum in the plants treated with the NPK biofertilizer (2.59 mg quercetin g−1 DM) and then the K biofertilizer (2.56 mg quercetin g−1 DM), but the N and P biofertilizers did not differ from the control (2.27 mg quercetin g−1 DM) significantly (Fig. 5). Since silymarin, the active ingredient of milk thistle, is a flavonoid compound composed of five flavonolignans, it can be concluded that the application of NPK and K biofertilizers increased silymarin content. Also, studies on the relationship between the accumulation of flavonolignans and the vegetative traits of milk thistle revealed a positive significant relationship between the accumulation of these compounds and plant height. Similarly, the increase in plant height and flavonoid content in the plants treated with the NPK biofertilizer in the present study supports it.Fig. 5The results of means comparison for the simple effect of biofertilizer on total flavonoid content. Note: N(b): nitrogen bio-fertilizer, P(b): phosphate bio-fertilizer, K(b): potash bio-fertilizer, NPK(b): complete bio-fertilizers.Full size imageDPPH radical scavengingAccording to the results of the means comparison in Fig. 6a, the capacity of inhibiting DPPH radical was increased by almost 5% under the no-irrigation conditions. Also, the results showed that under the full irrigation conditions, this capacity did not vary among the fertilizer treatments significantly. However, under the no-irrigation conditions, the treatments of K (58.8%), NPK (57.6%), and P biofertilizers (55.7%) had the highest inhibiting capacity and they were in the same statistical group, the N biofertilizer (51.6%) was in the intermediate group, and no-biofertilizer application (44.3%) exhibited the lowest DPPH radical scavenging capacity. The difference in antioxidant activity may be associated with the difference in phenol content and other active compounds. The percent scavenging of DPPH radical in thyme was measured by Mehran et al.40 to be 66.6%. The results of a study showed that drought stress increased DPPH free radicals scavenging41. Studies have generally reported the positive effect of biofertilizer on increasing antioxidant properties, e.g. DPPH free radicals scavenging42.Superoxide radical scavengingSuperoxide radical scavenging was higher under the full irrigation conditions (77.2%) than the no-irrigation conditions (72.3%). Also, among the biofertilizer treatments, it was the highest (81.4%) in the plants treated with the NPK biofertilizer followed by those treated with the K biofertilizer (79.7%). The plants treated with the P biofertilizer were at an intermediate level (75.3%), and those treated with the N biofertilizer (70.3%) and those not treated at all (70.0%) had the lowest scavenging capacity (Fig. 6b). Superoxide anion (O2−) is a reduced form of molecular oxygen that is a free radical composed of the electron transport systems of mitochondria. Some electrons that pass through the mitochondria chain reaction react with oxygen directly and form superoxide anion. Free radicals are highly reactive and mostly damage proteins and break down DNA strings43. With respect to the effect of biofertilizers on superoxide radical scavenging, it has been shown that biofertilizers increase this antioxidant trait42.Nitric oxide radical scavengingBased on the comparison of the means (Fig. 6c), the full irrigation conditions had higher nitric oxide radical scavenging (26.9%) than the no-irrigation conditions (27.3%). In addition, the trend of nitric oxide radical scavenging capacity for different biofertilizers (Fig. 6c) was similar to that of superoxide radical scavenging capacity. Nitric oxide radical scavenging capacity was the highest in the plants treated with the K biofertilizer (28.1%) and the lowest in those not treated with a biofertilizer (26.2%). Except for the NPK biofertilizer, all treatments were in the same statistical group with the control. In general, the bio-fertilizer treatment with full irrigation and without irrigation increased DPPH radical scavenging by about 7 and 25%, respectively, compared to the control treatment. Marcocci et al.44 concluded that nitric oxide radical scavengers compete with conducting oxygen to reduce nitrite oxide generation not only in the plants of the Lamiaceae but also in other plant species.Fig. 6The results of means comparison for the simple effect of irrigation and biofertilizer on DPPH radical scavenging (a), superoxide radical scavenging (b) and nitric oxide radical scavenging (c). Note: N(b): nitrogen bio-fertilizer, P(b): phosphate bio-fertilizer, K(b): potash bio-fertilizer, NPK(b): complete bio-fertilizers.Full size imageChain-breaking activityThe treatments of the NPK (16.0%), K (15.8%), and P biofertilizers (15.7%) had the highest chain-breaking activity with slight differences, respectively. They were in the same statistical group. But, the treatments of the N biofertilizer (13.6%) and no biofertilizer (13.0%) had the minimum chain breaking and were placed in the same statistical group (Fig. 7). In a study on the effect of humic acid on the antioxidant properties of thyme under the ecological conditions of Urmia, Iran, Taghipour et al.45 reported that the highest total phenol content, DPPH radical scavenging percentage, and chain-breaking activity were obtained in the first harvest from the application of humic acid and the highest DPPH radical scavenging percentage and chain-breaking activity were obtained in the second harvest from the treatment of humic acid.Fig. 7The results of means comparison for the simple effect of biofertilizer on chain-breaking activity. Note: N(b): nitrogen bio-fertilizer, P(b): phosphate bio-fertilizer, K(b): potash bio-fertilizer, NPK(b): complete bio-fertilizers.Full size imageMaterials and methodsThe present study was carried out at the agricultural research station of Miandoab city located in the south of West Azarbaijan province in Iran (Lat. 36°58’ N., Long. 46°06’ E., Alt. 1314 m.) during 2017. The climate of the Miandoab region is relatively hot in the summer, and relatively cold in the winter. The average rainfall in the region amounts to 289 mm. Table 3 contains meteorological parameters information related to the months of the experiment. To determine its physical and chemical characteristics and estimate fertilizer requirements46,47, the soil at the study site was randomly sampled from a depth of 0–30 cm before sowing. Then, they were mixed and sent to a laboratory. The results are presented in Table 4.Table 3 Meteorological information related to the months of the experiment in the crop year.Full size tableTable 4 Physicochemical properties of soil at the study site.Full size tableThe field trial was carried out a split-plot experiment based on a randomized complete block design with 10 treatments and 3 replications. The main plot was assigned to irrigation at two levels (I1: irrigation water with crop water requirement (CWR) of 100% and I2: no-irrigation conditions) and the sub-plot was assigned to bio-fertilizer at five levels (control, nitrogen bio-fertilizer- N(b), phosphate bio-fertilizer- P(b), potash bio-fertilizer- K(b), and complete bio-fertilizers- NPK(b)). In this study, the irrigation technique employed was the surface irrigation (furrow method) three irrigations with a water depth of 100 mm were conducted in the sensitive areas of the milk thistle plant, including the vegetative period, the pre-flowering period, and the seed filling period. As a result, the water requirement of this plant is 300 mm or 3000 m3 per ha. On May 15, May 30, and June 15, irrigation was done with an interval of 15 days. Furthermore, the growth period for this plant was four months (late March to mid-July). It should also be mentioned that the milk thistle plant from the research farm of Urmia University named after Dr. Amir Rahimi has been registered in the herbarium of the Urmia Agricultural Research Centre of West Azerbaijan Province with the international code 11,071-WESTA. In general, previous research experiments have shown that the amount of heavy elements in agricultural soils in the current research area is not too critical48,49.Azotobacter 1 contains the bacteria of the O4 strain of Azotobacter vinelandii, which fixes atmospheric N actively into the forms that are absorbable by plants. One 100-g container of Azotobacter 1 can be an effective replacement for 30–50 kg chemical N fertilizer. The phosphate biofertilizer contained two phosphate-solubilizing bacteria that decompose insoluble phosphorus compounds of soil by two mechanisms – the secretion of organic acids and enzyme phosphatase. Then, this nutrient becomes available to plants. Based on the amount of soil absorbable phosphorus, each package of this biofertilizer can replace 50–100% of the chemical phosphate fertilizer demand of plants. The biofertilizer Pota-Barvar-2 contains two potassium solubilizing bacteria that decompose insoluble potassium in the root zone and release its ions, thereby optimizing potassium uptake. So, it can be a replacement for at least 50% of potassium chemical fertilizers.The biofertilizers used in the study were applied to the seeds during sowing, for which the biofertilizers were diluted to the amount required to dampen the seeds for 1 ha. Then, the seeds were spread on a piece of plastic or a clean surface, and the diluted solution was applied to them with a sprayer. Then, the seeds were mixed with the solutions well. The seeds used in the experiment were from the local landrace of Western Europe, which has been domesticated and is extensively produced at the farms of this region. After a tillage operation in the autumn and land preparation in February 2017, a total of 20 plots were built, each with an area of 6 m2. The seeds were sown on rows with an inter-row spacing of 60 cm and an on-row spacing of 25 cm. They were sown at a depth of 3–5 cm in Late-March, 2017 after inoculation with the biofertilizers. After the germination and growth of the plants, the extra plants were thinned at the 2-4-leaf stage and the empty parts of the plots were re-planted on April 10, 2017. At the same time, the weeds were removed by hand. Each plot contained five sowing rows spaced by 2 m.It should be noted that in the full irrigation treatment, the irrigation of the plots was initiated one month after sowing and repeated every 15 days. Also, the plants were sown at the physiological maturity stage four months after sowing. Since the maturity of the plants and plant constituent parts was not concurrent, the harvest was performed at several stages after the drying and yellowing of the plants. The harvested plants were sun-dried for several days until they lost their moisture. Then, the plants of each plot were weighed and crushed to have their seeds separated. Then, the remaining foliage was separated from the seeds by screening and airing and the crop of each plot was poured into separate packages and weighed.To determine morphological traits and yield components, 10 plants were randomly harvested from each plot to measure the traits like plant height, crown diameter, capitule number, and the number of auxiliary branches. Also, to determine seed yield, two marginal rows and 0.5 m from each side of the plots were eliminated as the marginal effect and the plants of the remaining area were harvested to find out their seed yield. The ratio of the economical part (seed) to total dry weight (biological yield) was taken as the seed harvest index. Indeed, the harvest index was calculated by the following equation and expressed in percent:$${text{Harvest index }}={text{ }}frac{{{text{Seed yield}}}}{{{text{Biological yield}}}}$$
(1)
To find out the oil yield, the ground seeds were oven-dried at 75 °C for 24 h. Then, 2.5 g of each sample was wire-wrapped in a piece of thin cloth and was then placed in an oil extraction device. The samples were boiled in 300 cc of n-hexane for 6 h. Then, the seeds wrapped in the cloth were oven-dried again at 75 °C for 24 h. Then, its weight was placed in the equation of oil calculation to determine the oil percentage. Afterward, the oil samples were poured into micro-tubes and were sent to a laboratory to be analyzed. Oil yield per plant was calculated by the following equation:$${text{Oil yield }}={text{ Seed yield per plant }} times {text{ Oil percent}}$$
(2)
To estimate the flavonoid content of different parts, 10 µL of the plant extract was first diluted with 1 mL of distilled water and it was then added with 0.075 mL of sodium nitrite (5%). Five minutes after the reaction, 0.15 mL of aluminum chloride (10%) was added, and 6 min after the reaction, 0.5 mL of sodium hydroxide (1 mol L−1) was incorporated and was adjusted to a final volume of 3 mL. Finally, its absorption was read at 510 nm and the total flavonoid content was determined by the quercetin standard curve. To find out DPPH stable radical scavenging rate, firstly 10 µL of the extract was mixed with 2 mL of methanol solution (0.004%). Then, the absorption of the solution was read at 517 nm after 30 min of incubation (at the darkness at room temperature). DPPH scavenging activity was calculated by the following Eqs50,51.:$${text{Percent DPPH radical suppression }}={text{ }}frac{{{{text{A}}_{{text{blank}}}}{text{ – }}{{text{A}}_{{text{sample}}}}}}{{{{text{A}}_{{text{blank}}}}}} times 100$$
(3)
In which Ablank represents the extract-free reaction mixture absorption and Asample represents the extract-containing reaction mixture absorption.Superoxide free radical scavenging was measured by the procedure described in Beauchamp and Fridovich52. So, 9 mL of tris-HCL buffer solution (pH = 8.2, 50 mmol L−1) was poured into a test tube and it was placed in a water bath at 25 °C for 20 min. Then, 40 µL of pyrogallol solution (45 mmol L−1 pyrogallol in 10 mmol L−1 hydrochloric acid), already incubated at 25 °C, was injected into the upper part of the test tube with a µL syringe, and they were mixed. The solution was incubated at 25 °C for 3 min, and immediately after that, one drop of ascorbic acid was poured into the mixture to stop the reaction. The mixture absorbance was read at 320 nm as A0 after 5 min. A0 represents the pyrogallol auto-oxidation rate. The auto-oxidation rate A1 was calculated by the same procedure with the only difference being the addition of 50 µL of tris buffer to the extract. At the same time, a control blank of the reaction compound was considered as A2. Percent radical scavenging was calculated by the following equation:$${text{Percent scavening of superoxide radicals}}=frac{{{A_0} – ({A_1} – {A_2})}}{{{A_0}}} times 100$$
(4)
To collect free nitric radicals, 10 µL of the extract was added with 0.5 mL of phosphate-buffered saline (10 mmol) and 2 mL of sodium nitroprusside (10 mmol). It was, then, incubated at 25 °C for 150 min. Then, 0.5 mL of this solution was mixed with 1 mL of sulfanilic acid (0.33% in glacier acetic acid 10%) and was rested for 5 min for the reaction to complete. Then, 1 mL of naphthyl ethylene diamine dihydrochloride (0.1%) was added and the mixture was kept at 25 °C for 30 min during which a pink color formed in the solution. Next, the absorbance was read at 540 nm to determine percent inhibition by the following Eqs44,53.:$${text{Percent scavenging of free nitric oxide radicals}}=frac{{{{text{A}}_{{text{blank}}}}{text{ – }}{{text{A}}_{{text{sample}}}}}}{{{{text{A}}_{{text{sample}}}}}} times 100$$
(5)
In which Ablank represents the extract-free reaction mixture absorbance and Asample represents the extract-containing reaction mixture absorbance.Chain breaking activity was measured by the DPPH reagent and the method described in Brand-Williams et al.54 with slight modifications. So, 10 µL of the extract was mixed with 1.9 mL of methanol solution (0.004%) of DPPH. Then, its absorbance was read at time zero and again 30 min after incubation at the darkness and room temperature at 515 nm. The reaction speed was calculated by the following equation:$$Abs – 3 – Abs0 – 3= – 3kt$$
(6)
There were more than two treatments groups in the present study. Therefore, an analysis of variance was performed to investigate the presence or absence of treatment differences overall. If treatment differences were significant, a comparison of means was performed based on Duncan’s test. The test of normality of experimental error distribution and the analysis of variance (ANOVA) for all traits were performed in the SAS 9.4 software package. The comparison of the means for all traits was carried out by Duncan’s multiple range test at the P < 0.05 probability level. Finally, all graphs were drawn in MS-Excel.ConclusionsThe milk thistle is of high importance due to its drought stress adaptation and resistance and nowadays the use of biofertilizer to curb on the use of chemical fertilizer and to increase crop yields is important for the movement towards sustainable agriculture, which can be much more important under no-irrigation conditions and since little research has addressed the effect of combined use of no-irrigation conditions and biofertilizers on medicinal plants including milk thistle, the present study focused on the effect of bio-fertilizers on the quality and quantity of thistle under no-irrigation conditions. Bio-fertilizers effectively increase the plant’s tolerance to drought stress, prevent a reduction in grain yield. In the present study, bio-fertilizers were examined to determine whether they influence the plant’s tolerance to drought stress and whether, on the other hand, the yield of the plant is reduced.The results showed that most morphological and biochemical traits of milk thistle were affected by the irrigation conditions and biofertilizers. The use of biofertilizers improved these traits versus the control. Given the significant role of biofertilizers in the development of sustainable agriculture, they can be a good candidate in case there is a need for nutrient supplements for this plant. Under drought stress conditions, the combination of bio-fertilizers (NPK(b)) and irrigation helps the plant to tolerate drought stress easily and also improves the plant’s antioxidant properties (phenolic and flavonoid compounds). Although the absence of irrigation results in a decrease in milk thistle yield, the utilization of bio-fertilizers, specifically complete bio-fertilizer and phosphate, can effectively compensate for the decrease in yield by enhancing nutritional conditions. It is recommended that the milk thistle plant be considered for cultivation and exploitation in arid and semi-arid areas and rain-fed areas. In addition to its medicinal properties, it is recommended to utilize the milk thistle plant in the food industry and animal husbandry.

Data availability

The datasets generated and analyses during the current study are not publicly available due to the work on the data and subsequent studies, but are available from the corresponding author on reasonable request.
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Download referencesAcknowledgementsThe authors are thankful to the Office of Vice Chancellor for Research and Technology, Urmia University. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Author informationAuthors and AffiliationsDepartment of Plant Production and Genetics, Faculty of Agriculture, Urmia University, Urmia, IranJafar Ghaffarzadeh, Reza Amirnia & Amir RahimiAgricultural Engineering Research Department, West Azerbaijan Agricultural and Natural Resources Research and Education Center, AREEO, Urmia, IranAfshin KhorsandAuthorsJafar GhaffarzadehView author publicationsSearch author on:PubMed Google ScholarReza AmirniaView author publicationsSearch author on:PubMed Google ScholarAmir RahimiView author publicationsSearch author on:PubMed Google ScholarAfshin KhorsandView author publicationsSearch author on:PubMed Google ScholarContributionsJ.G. conceived the idea and wrote the manuscript. R.A. and A.R. reviewed the collected data, and prepared the equipment and materials. J.G. and A.K. was responsible for editing, original data and text preparation. All authors took responsibility for the integrity of the data that is present in this study.Corresponding authorCorrespondence to
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Reprints and permissionsAbout this articleCite this articleGhaffarzadeh, J., Amirnia, R., Rahimi, A. et al. Effect of irrigation and bio-fertilizers on morphological and bio-chemical traits of milk thistle.
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AbstractThe science of metabolic profiling exploits chemical compound byproducts of metabolism called metabolites1 that explain internal biological functions, physiological health and disease, and provide evidence of external influences specific to an organism’s habitat. Here we assess palaeometabolomes from fossilized mammalian hard tissues as a molecular ecological strategy to provide evidence of an ancient organism’s relationship with its environment. From eastern, central and southern African Plio-Pleistocene localities of palaeoanthropological significance, we study six fossils from Olduvai Gorge, Tanzania, one from the Chiwondo Beds, Malawi, and one from Makapansgat, South Africa. We perform endogeneity assessments by analysing palaeometabolomes of palaeosols and the effects of owl digestion on rodent bones to enable prudent ecological inferences. Diagenesis is indicated by metabolites of collagenase-producing bacteria2, whereas the preservation of peptides including those of collagen are identified by proteomics. Endogenous metabolites document biological functions and exogenous metabolites render environmental details including soil characteristics and woody cover, and enable annual minimum and maximum rainfall and temperature reconstructions at Olduvai Gorge, supporting the freshwater woodland and grasslands of Olduvai Gorge Bed I3,4,5, and the dry woodlands and marsh of Olduvai Gorge Upper Bed II6. All sites denote wetter and/or warmer conditions than today. We infer that metabolites preserved in hard tissues derive from an extravasated vasculature serum filtrate that becomes entombed within developing mineralized matrices, and most probably survive palaeontological timeframes in the nanoscopic ‘pool’ of structural-bound water that occurs in hard tissue niches7.

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Fig. 1: Representative images of fossil specimens.Fig. 2: PLS-DA of the top 100 most variable metabolites.Fig. 3: Bone ultrastructure and metabolite niches.Fig. 4: Average metabolite molecular masses of extant and fossil hard tissues.

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Data availability

The raw mass spectrometry metabolomics data generated during this study are available at MassIVE (UCSD) (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) under the deposition number MSV000097146. Proteomics LC–MS data (proteomics raw mass spectrometry data, peak lists and results) that support the findings of this study are deposited to the ProteomeXchange Consortium via the MassIVE partner repository and can be retrieved with the accession code MSV000097173 and with the dataset identifier PXD061016.
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Download referencesAcknowledgementsFunding for this project was provided by The Leakey Foundation (grant number Spring202310420) to T.G.B. We thank all institutions, which provided and supported sampling: the CMCK Karonga, Malawi, NMHN Paris, France, and the Senckenberg Research Institute and the Natural History Museum Frankfurt, Germany. We express our gratitude to the Werner Reimers Foundation in Bad Homburg, Germany, which provides the Gustav Heinrich Ralph von Koenigswald collection as a permanent loan for scientific research to the Senckenberg Research Institute and Natural History Museum Frankfurt. Thanks go to the New York University Grossman School of Medicine’s Applied Bioinformatics Laboratories for access to QIAGEN IPA. The Zeiss Gemini 300 FE-SEM used for evaluating bone microanatomy was provided courtesy of the National Institutes of Health S10 Shared Instrumentation Program, grant number 1S10OD026989-01; mass spectrometry instrumentation, National Institutes of Health grant numbers S10 OD023659 and S10 RR027990. We thank P. Ausili and L. Kaleel for assistance with metabolite annotations and data organization. N.R. and M.D.M. are members of the Fonds de Recherche du Québec–Santé (FRQS) Centre for Structural Biology Research at McGill University, and the FRQS Network for Intersectorial Research in Sustainable Oral and Bone Health. M.D.M. is the Canada Research Chair in Biomineralisation, and N.R. is a William Dawson Scholar at McGill University. A.S. is supported by the French government in the framework of the University of Bordeaux’s IdEx ‘Investments for the Future’ programme/GPR ‘Human Past’. This project is an outcome of the 2010 Max Planck Research Award to T.G.B., endowed by the German Federal Ministry of Education and Research to the Max Planck Society and the Alexander von Humboldt Foundation in respect of the Hard Tissue Research Program in Human Paleobiomics.Author informationAuthors and AffiliationsDepartment of Molecular Pathobiology, New York University College of Dentistry, New York, NY, USATimothy G. Bromage, Bin Hu & Shoshana YakarDepartment of Anthropology, New York University, New York, NY, USATimothy G. BromageSenckenberg Research Institute and Natural History Museum, Frankfurt am Main, GermanyTimothy G. Bromage, Ottmar Kullmer, Friedemann Schrenk & Jülide KubatInstitute of Systematics and Evolution of Biodiversity, National Museum of Natural History, Paris, FranceChristiane DenysDepartment of Neuroscience and Physiology and Neuroscience Institute, NYU Grossman School of Medicine, New York, NY, USAChristopher Lawrence De Jesus, Hediye Erdjument-Bromage & Thomas A. NeubertInstitute of Ecology, Evolution and Diversity, Goethe University, Frankfurt am Main, GermanyOttmar Kullmer & Friedemann SchrenkEarth and Life History, Hessisches Landesmuseum Darmstadt, Darmstadt, GermanyOliver SandrockFaculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, Quebec, CanadaMarc D. McKee, Natalie Reznikov & Daniel J. BussDepartment of Anatomy and Cell Biology, McGill University, Montreal, Quebec, CanadaMarc D. McKee & Natalie ReznikovDepartment of Bioengineering, McGill University, Montreal, Quebec, CanadaNatalie Reznikov & Eran IttahDepartment of Earth and Planetary Sciences, Rutgers, the State University of New Jersey, New Brunswick, NJ, USAGail M. AshleyEurofins Lancaster Laboratories PSS, Lancaster, PA, USASher B. Poudel & Antoine SouronUniversité de Bordeaux, CNRS, Ministère de la Culture, PACEA, UMR 5199, Pessac, FranceDaniel J. BussDepartment of Dental Hygiene and Dental Assisting, New York University College of Dentistry, New York, NY, USASasan RabiehAuthorsTimothy G. BromageView author publicationsSearch author on:PubMed Google ScholarChristiane DenysView author publicationsSearch author on:PubMed Google ScholarChristopher Lawrence De JesusView author publicationsSearch author on:PubMed Google ScholarHediye Erdjument-BromageView author publicationsSearch author on:PubMed Google ScholarOttmar KullmerView author publicationsSearch author on:PubMed Google ScholarOliver SandrockView author publicationsSearch author on:PubMed Google ScholarFriedemann SchrenkView author publicationsSearch author on:PubMed Google ScholarMarc D. McKeeView author publicationsSearch author on:PubMed Google ScholarNatalie ReznikovView author publicationsSearch author on:PubMed Google ScholarGail M. AshleyView author publicationsSearch author on:PubMed Google ScholarBin HuView author publicationsSearch author on:PubMed Google ScholarSher B. PoudelView author publicationsSearch author on:PubMed Google ScholarAntoine SouronView author publicationsSearch author on:PubMed Google ScholarDaniel J. BussView author publicationsSearch author on:PubMed Google ScholarEran IttahView author publicationsSearch author on:PubMed Google ScholarJülide KubatView author publicationsSearch author on:PubMed Google ScholarSasan RabiehView author publicationsSearch author on:PubMed Google ScholarShoshana YakarView author publicationsSearch author on:PubMed Google ScholarThomas A. NeubertView author publicationsSearch author on:PubMed Google ScholarContributionsT.G.B. conceived the study and led the work. C.D., J.K., O.K., F.S. and O.S. prepared and provided fossil samples and extant representatives of African species. G.M.A. and C.D. provided palaeosols. S.Y. and S.B.P. prepared and provided the extant laboratory mouse material, A.S. provided likelihoods of plant resources based on isotopic evidence. B.H. undertook the histology and light and electron microscopy. S.R. produced the fossil bone extracts. T.A.N., C.L.D.J. and H.E.-B. performed the metabolomics and proteomics and their relevant bioinformatics. M.D.M., N.R., D.J.B. and E.I. elaborated the interpretation of the bone ultrastructural metabolite niches. T.G.B. and S.R. annotated the metabolite lists from online databases, and T.G.B. interpreted the metabolic and ecological profiles and wrote the manuscript, with valuable contributions from all co-authors.Corresponding authorCorrespondence to
Timothy G. Bromage.Ethics declarations

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Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended data figures and tablesExtended Data Fig. 1 Polarised light images of specimen M-D. Mus sp. or Dendromus sp., Bed I, Level FLKN1 M3.a. Birefringent brightness at top and bottom is attributed to heavily crystalised bone domains, while the central curvilinear arc of brightness is attributed to collagen. FW = 1.547 mm; b) Detail of bright bone lamellae and osteocyte lacunae interspersed among them. FW = 0.324 mm.Extended Data Fig. 2 Polarised light image of specimen Sm. Saccostomus cf. mearnsi, Bed I, Level FLKN1.a. Birefringent brightness is attributed to collagen. FW = 1.343 mm; b) Detail of bright domains and osteocyte lacunae interspersed among them. FW = 0.324 mm.Extended Data Fig. 3 Polarised light image of specimen Gg. Gerbilliscus gentryi, Bed I, Level FLKN1 M1.a. Birefringent brightness is attributed to collagen. FW = 3.922 mm; b) Detail of bright domains and osteocyte lacunae interspersed among them. FW = 0.324 mm.Extended Data Fig. 4 Polarised light image of specimen Gi. Gerbilliscus sp. indet, Bed I, Level DK.a. Birefringent brightness is attributed to collagen. FW = 4.506 mm; b) Detail of bright domains and osteocyte lacunae interspersed among them. FW = 0.324 mm.Extended Data Fig. 5 Polarised light image of specimen Xi. Xerus cf. inauris, Bed I, Level FLKN1 M3.a. Birefringent brightness is attributed to collagen. FW = 4.434 mm; b) Detail of bright domains and osteocyte lacunae interspersed among them. FW = 0.324 mm.Extended Data Fig. 6 BSE-SEM image of specimen M-D. Mus sp. or Dendromus sp., Bed I, Level FLKN1 M3.a. Macroscopic view of the bone fragment. FW = 1.515 mm; b. Detail of bone and osteocyte lacunae and their associated canaliculi. Large cracks are due to diagenesis or preparation. FW = 0.25 mm.Extended Data Fig. 7 BSE-SEM image of specimen Sm. Saccostomus cf. mearnsi, Bed I, Level FLKN1.a. Macroscopic view of the bone fragment. FW = 1.329 mm; b. Detail of bone illustrating diagenetically disorganized bony structure but preserving some vascular canals and osteocyte lacunae. FW = 0.25 mm.Extended Data Fig. 8 BSE-SEM image of specimen Gg. Gerbilliscus gentryi, Bed I, Level FLKN1 M1.a. Macroscopic view of the bone fragment. FW = 3.726 mm; b. Detail of bone illustrating a vascular canal, mineralization density variation, and osteocyte lacunae and their associated canaliculi. FW = 0.25 mm.Extended Data Fig. 9 BSE-SEM image of specimen Gi. Gerbilliscus sp. indet, Bed I, Level DK.a. Macroscopic view of the bone fragment. FW = 4.261 mm; b. Detail of bone illustrating a vascular canal (bottom), mineralization density variation, and osteocyte lacunae and their associated canaliculi. Large cracks are due to diagenesis or preparation. FW = 0.25 mm.Extended Data Fig. 10 BSE-SEM image of specimen Xi, Xerus cf. inauris, Bed I, Level FLKN1 M3.a. Macroscopic view of the bone fragment. FW = 4.211 mm; b. Detail of bone illustrating a vascular canal, mineralization density variation, and osteocyte lacunae and their associated canaliculi. FW = 0.25 mm.Supplementary informationSupplementary InformationSupplementary Figs 1–10.Reporting SummarySupplementary Table 1Detailed list of samples.Supplementary Table 2Calcium/phosphate ratios from the study sample.Supplementary Table 3UM-HET3 and C57BL-6J mice and diets.Supplementary Table 4Olduvai Gorge Mus sp. or Dendromus sp., extant MNHN CD, palaeosols.Supplementary Table 5Olduvai Gorge Saccostomus cf. mearnsi, extant MNHN 1991-817, palaeosols.Supplementary Table 6Olduvai Gorge Gerbilliscus gentryi, extant MNHN NC, palaeosols.Supplementary Table 7Olduvai Gorge Gerbilliscus sp. indet, MNHN NC, palaeosols.Supplementary Table 8Olduvai Gorge Xerus cf. inauris, MNHN 2005-575, palaeosols.Supplementary Table 9Olduvai Gorge, Kolpochoerus majus, von Koenigswald 1854.Supplementary Table 10Chiwondo Beds, Elephas recki shungurensis, EE, palaeosol HCRP-RC-11.Supplementary Table 11Makapansgat, Bovidae, EW, palaeosol TF12.Supplementary Table 12Site metabolite comparisons.Supplementary Table 13OG soil carbonates.Supplementary Table 14Statistical tests.Supplementary Table 15Fossil Bone Proteomics.Supplementary Table 16Summary statistics.Rights and permissionsSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Reprints and permissionsAbout this articleCite this articleBromage, T.G., Denys, C., De Jesus, C.L. et al. Palaeometabolomes yield biological and ecological profiles at early human sites.
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