Promoting the Growth of Pinus sylvestris var. mongolica Seedlings and Improving Rhizosphere Fungal Community Structure through Interaction between Trichoderma and Ectomycorrhizal Fungi

Saiyaremu Halifu (Agricultural College of Shihezi University, Shihezi, China;School of Forestry, Northeast Forestry University, Harbin, China)
Xun Deng (Institute of Forestry Protection, Heilongjiang Forestry Academy, Harbin, China)
Jun Zhang (Shandong Province Key Laboratory of Eco-Environmental Science for Yellow River Delta, Binzhou University,Binzhou, 256600, China)
Jiangbao Xia (Shandong Province Key Laboratory of Eco-Environmental Science for Yellow River Delta, Binzhou University,Binzhou, 256600, China)
Xiaoshuang Song (Institute of Forestry Protection, Heilongjiang Forestry Academy, Harbin, China)
Ruiqing Song (School of Forestry, Northeast Forestry University, Harbin, China)

Abstract


In this study, pot experiments were conducted on the seedlings of Pinus sylvestris var. mongolica to study the influence of Trichoderma (Trichoderma harzianum E15) and Ectomycorrhizal fungi (Suillus luteus N94) on the growth of these seedlings. In particular, the effects of these fungi on the fungal community structure in the rhizosphere soil of the seedlings were investigated. Inoculation with Trichoderma harzianum E15 and Suillus luteus N94 significantly (P < 0.05) promoted the growth of the Pinus sylvestris seedlings. The non-metric multidimensional scaling (NMDS) results indicated a significant difference (P < 0.05) between the fungal community structures in the rhizosphere soil of the annual and biennial seedlings. In the rhizosphere soil of annual seedlings, the main fungi were Ascomycota, Basidiomycota, Zygomycota. Ascomycota, Basidiomycota, Mortierellomycota, and p-unclassified-k-Fungi were the main fungi in the rhizosphere soil of biennial seedlings. The dominant genus in the rhizosphere soil and a key factor promoting the growth of the annual and the biennial seedlings was Trichoderma, Suillus, respectively. Both of them were negatively correlated with the relative abundance of microbial flora in the symbiotic environment. Trichoderma had a significant promoting effect on the conversion of total phosphorus, total nitrogen, ammonium nitrogen, nitrate nitrogen, and the organic matter in the rhizosphere soil of the seedlings, while Suillus significantly promoted the conversion of organic matter and total phosphorus.


Keywords


Pinus sylvestris var. mongolica; Growth promotion; Rhizosphere fungal community; Trichoderma harzianum; Suillus luteus

Full Text:

PDF

References


[1]Qi JY, Yin DC, Song RQ. Effects of inoculating Suilusleutus and Trichoderma virens on damping-off resistance of Mongolian Pine seedlings. Journal of Jilin Agricultural University, 2018, 40, 43-49.

[2]Lamichhane JR, Dürr Carolyne, Schwanck André, A et al. Integrated management of damping-off diseases. A review. Agronomy for Sustainable Development. 2017, 37, 10. DOI: 10.1007/s13593-017-0417-y.

[3]John CJ, Jishma P, Karthika NR, et al. Pseudomonas fluorescens R68 assisted enhancement in growth and fertilizer utilization of Amaranthus tricolor (L.). Biotech. 2017, 7, 256. DOI: 10.1007/s13205-017-0887-2.

[4]Zhang FG, He YQ, Cobb AB, et al. Trichoderma biofertilizer links to altered soil chemistry, altered microbial communities, and improved grassland biomass. Frontiers in Microbiology. 2018, 9, 848. DOI: 10.3389/fmicb.2018.00848.

[5]Li Y, Chen Z, He JZ, et al. Ectomycorrhizal fungi inoculation alleviates simulated acid rain effects on soil ammonia oxidizers and denitrifiers in Masson pine forest. Environmental Microbiology. 2018, 29. DOI: 10.1111/1462-2920.14457.

[6]Perrig D, Boiero ML, Masciarelli OA, et al. Plant-growthpromoting compounds produced by two agronomically important strains of Azospirillumbrasilense, and implications for inoculant formulation. Applied Microbiology & Biotechnology. 2007, 75, 1142- 1150. DOI: 10.1007/s00253-007-0909-9.

[7]Irum N, Asghari B, Hassan TU. Isolation of phytohormones producing plant growth promoting rhizobacteria from weeds growing in Khewra salt range, Pakistan and their implication in providing salt tolerance to Glycine max L. African Journal of Biotechnology. 2009, 8. DOI: 10.5897/AJB09.1176.

[8]Yasar E, Sezai E, Ayhan H, et al. Effects of plant growth promoting rhizobacteria (PGPR) on rooting and root growth of kiwifruit (Actinidia deliciosa) stem cuttings. Biological Research. 2010, 43. DOI: 10.4067/S0716-97602010000100011.

[9]Smith SE, Jakobsen I, Gronlund M, et al. Roles of arbuscular mycorrhizas in plant phosphorus nutrition: interactions between pathways of phosphorus uptake in arbuscular mycorrhizal roots have important implications for understanding and manipulating plant phosphorus acquisition. Plant Physiology. 2011, 156, 1050-1057. DOI: 10.1104/pp.111.174581.

[10]Contreras-Cornejo HA, Macías-Rodríguez L, AlfaroCuevas R, et al. Trichoderma spp. improve growth of Arabidopsis seedlings under salt stress through enhanced root development, osmolite production, and Na+ elimination through root exudates. Molecular Plant-Microbe Interactions. 2014, 27, 503-514. DOI: 10.1094/MPMI-09-13-0265-R. DOI: 10.1073/ pnas.1322671111.

[11]Eugenia Morán-Diez, Belén R, Sara D, et al. Transcriptomic response of Arabidopsis thaliana after 24 h incubation with the biocontrol fungus Trichoderma harzianum. Journal of plant physiology. 2012, 169, 600-620. DOI: 10.1016/j.jplph.2011.12.016.

[12]Plett JM, Daguerre Y, Wittulsky S, et al. Effector MiSSP7 of the mutualistic fungus Laccaria bicolor stabilizes the Populus JAZ6 protein and represses jasmonic acid (JA) responsive genes. Proceedings of the National Academy of Sciences, 2014, 111, 8299.

[13]Behie SW, Bidochka MJ. Nutrient transfer in plantfungal symbioses. Trends in Plant Science. 2014, 19, 734-740. DOI: 10.1016/j.tplants.2014.06.007.

[14]Sana K, Izzah S, Baig DN, et al. Contribution of zinc solubilizing bacteria in growth promotion and zinc content of wheat. Frontiers in Microbiology. 2017, 8, 2593. DOI: 10.3389/fmicb.2017. 02593.

[15]De SA, Quintero JM, Avilés M, et al. Effect of Trichoderma asperellum strain T34 on iron nutrition in white lupin. Soil Biology and Biochemistry, 2009, 41, 2453-2459.

[16]Contreras-Cornejo HA, Macías-Rodríguez Lourdes, Vergara AG, et al. Trichoderma modulates stomatal aperture and leaf transpiration through an abscisic acid-dependent mechanism in Arabidopsis. Journal of Plant Growth Regulation. 2015, 34, 425-432. DOI: 10.1007/s00344-014-9471-8.

[17]Frank B. Uber die auf Wurzelsymbiose beruhende Ernährung gewisser Bäume durch unterirdische Pilze. Berichte Der Deutschen Botanischen Gesellschaft, 1885, 3, 128-145.

[18]Smith SE, Read DJ. Mycorrhizal symbiosis (Third Edition). London: Academic press, 2008.

[19]Rousseau JVD, Reid CPP, English RJ. Relationship between biomass of the mycorrhizal fungus Pisolithus tinctorius and phosphorus uptake in loblolly pine seedlings. Soil Biology and Biochemistry. 1992, 24, 0-184. DOI: 10.1016/0038-0717(92)90276-4.

[20]Čatská V, Smith SE, Read DJ. Mycorrhizal symbiosis. Biologia Plantarum, 1997, 40. DOI: 10.1023/A:1000902213906.

[21]Harrier LA, Watson CA. The potential role of arbuscular mycorrhizal (AM) fungi in the bioprotection of plants against soil-borne pathogens in organic and/or other sustainable farming systems. Pest Management Science. 2004, 60, 149-157. DOI: 10.1002/ps.820.

[22]Yin DC, Deng X, Song RQ. Physiological responses of Pinus sylvestris var. mongolica seedlings to the interaction between Suillus grevillei N40 and Trichoderma virens T43. Chinese Journal of Ecology, 2014, 33, 2142-2147.

[23]Alfred OMO, Patrick OA. Soil fertility and crop productivity in African sustainable agriculture. Sustainable Agriculture Reviews. Springer International Publishing. 2015. DOI: 10.1007/978-3-319-09132-7_6.

[24]Yin DC, Deng X, Chet I, et al. Inhibiting effect and mechanism of Trichoderma virens T43 on four major species of forest pathogen. Chinese Journal of Ecology, 2014, 7,1911-1919.

[25]Yin DC, Song RQ, Qi JY, et al. Ectomycorrhizal fungus enhances drought tolerance of Pinus sylvestris var. mongolica seedlings and improves soil condition. Journal of Forestry Research. 2018, 29, 331-344.DOI: CNKI:SUN:LYYJ.0.2018-06-031.

[26]Deng X, Song XS, Song RQ. Effect of inoculating Phialocephala fortinii D575 and Suillus luteus N94 on the growth of Pinus sylvestris var. mongolica and its resistant to damping-off. Forest Pest and Disease. 2017, 36, 21-25.

[27]Collignon C, Uroz S, Turpault MP, et al. Seasons differently impact the structure of mineral weathering bacterial communities in beech and spruce stands. Soil Biology & Biochemistry. 2011, 43, 2012-2022. DOI: 10.1016/j.soilbio.2011.05.008.

[28]Hussain S, Zhang M, Zhu XX, Khan, et al. Significance of Fe(II) and environmental factors on carbon-fixing bacterial community in two paddy soils. Ecotoxicology and Environmental Safety. 2019,182: 109456. DOI: 10.1016/j.ecoenv.2019.109456.

[29]Halifu S, Deng X, Song XS, et al. Effects of Sphaeropsis blight on rhizosphere soil bacterial community structure and soil physicochemical properties of Pinus sylvestris var. mongolica in Zhanggutai, China. Forests. 2019,10:954. DOI: 10.3390/f10110954.

[30]Yang CX, Liu WZ, He ZW, et al. Freezing/thawing pretreatment coupled with biological process of thermophilic Geobacillus sp. G1: Acceleration on waste activated sludge hydrolysis and acidification. Bioresource Technology. 2015, 175, 509-516. DOI: 10.1016/j.biortech.2014.10.154.

[31]Buée M, Reich M, Murat C, et al. 454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity. New Phytologist. 2009, 184, 449- 456. DOI: 10.1111/j.1469-8137.2009.03003.x.

[32]Yong C, Jiang YM, Huang HY, et al. Long-term and high-concentration heavy-metal contamination strongly influences the microbiome and functional genes in Yellow River sediments. Science of the Total Environment. 2018, 637-638, 1400-1412. DOI: 10.1016/j.scitotenv.2018.05.109.

[33]Zhou J, Deng Y, Shen L, et al. Temperature mediates continental-scale diversity of microbes in forest soils. Nature Communications. 2016, 7, 12083. DOI: 10.1038/ncomms12083.

[34]Fu YJ, Zhang JL, Hou XQ. Comparative analysis of fungi diversity in rizospheric and non-rhizospheric soil from Cypripedium macranthum estimated via high-throughput sequencing. Acta Agriculturae Boreali-occidentalis Sinica. 2019,28, 1–7.

[35]Anne S, Jacquiod S, Vestergaard G, et al. Analysis of soil microbial communities based on amplicon sequencing of marker genes. Biology & Fertility of Soils. 2017, 1-5. DOI: 10.1007/s00374-017-1205-1.

[36]Magoč Tanja, Salzberg Steven L, Notes Author. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics. 2011, 27(21):2957-2963. DOI: 10.1093/bioinformatics/btr507.

[37]Bolger AM, Marc L, Bjoern U. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014, 30, 2114-2120. DOI: 10.1093/bioinformatics/btu170.

[38]Kljalg U, Nilsson HR, Abarenkov K, et al. Towards a unified paradigm for sequence-based identification of Fungi. Molecular Ecology. 2013, 22, 5271-5277. DOI: 10.1111/mec.12481.

[39]Washington HG. Diversity, biotic and similarity indices: A review with special relevance to aquatic ecosystems. Water Res. 1984, 18:653-694. DOI: 10.1016/0043-1354(84)90164-7.

[40]Dixon P. VEGAN, a package of R functions for community ecology. Journal of Vegetation Science. 2003, 14, 927-930. DOI: 10.1111/j.1654-1103.2003.tb02228.x.

[41]Xue L, Ren H, Brodribb TJ, et al. Long term effects of management practice intensification on soil microbial community structure and co-occurrence network in a non-timber plantation. Forest Ecology and Management. 2020, 459: 117805. DOI: 10.1016/j.foreco.2019.117805.

[42]Quan Y, Abdel A, Belkacem OB. Structural analysis for fault detection and isolation in fuel cell stack system[M]// Sustainability in Energy and Buildings. Springer Berlin Heidelberg. 2009. DOI: 10.1007/978-3-642-03454-1_25.

[43]Gentleman R, Huber W, Carey VJ. R Language[M]// International Encyclopedia of Statistical Science. Springer Berlin Heidelberg. 2011.

[44]Ben AM, Lopez D, Triki MA. et al. Beneficial effect of Trichoderma harzianum strain Ths97 in biocontrolling Fusarium solani causal agent of root rot disease in olive trees. Biological Control. 2017, 110,70-78. DOI: 10.1016/j.biocontrol.2017.04.008.

[45]López AC, Alvarenga AE, Zapata PD, et al. Trichoderma spp. from Misiones, Argentina: effective fungi to promote plant growth of the regional crop Ilex paraguariensis St. Hil. Mycology. 2019, 1-12. DOI: 10.1080/21501203.2019.1606860.

[46]Sharma R, Magotra A, Manhas RS, et al. Antagonistic potential of a psychrotrophic fungus: Trichoderma velutinum ACR-P1. Biological Control. 2017, 115, 12- 17.DOI: 10.1016/j.biocontrol.2017.08.024.

[47]Péret B, De Rybel B, Casimiro I, et al. Arabidopsis lateral root development: an emerging story. Trends in Plant Science. 2009, 14, 399-408. DOI: 10.1016/j.tplants.2009.05.002.

[48]Tsukaya H. Leaf morphogenesis: genetic regulations for length, width and size of leaves. Tanpakushitsu kakusan koso Protein Nucleic Acid Enzyme. 2002, 47(12 Suppl), 1576-1580.

[49]Albrecht T, Argueso CT. Should I fight or should I grow now? The role of cytokinins in plant growth and immunity and in the growth-defence trade-off. Annals of Botany. 2016, 119, 725-735. DOI: 10.1093/aob/mcw211.

[50]Giron D, Frago E, Gaëlle G, et al. Cytokinins as key regulators in plant-microbe-insect interactions: connecting plant growth and defence. Functional Ecology, 2013, 27, 599-609.

[51]Atkinson D, Black KE, Forbes PJ, et al. The influence of arbuscular mycorrhizal colonization and environment on root development in soil. European Journal of Soil Science. 2003, 54, 751-757. DOI: 10.1046/j.1351-0754.2003.0565.x.

[52]Lilleskov EA, Bruns TD, Dawson TE, et al. Water sources and controls on water‐loss rates of epigeous ectomycorrhizal fungal sporocarps during summer drought. New Phytologist. 2009, 182, 483-494. DOI: 10.1111/j.1469-8137.2009.02775.x.

[53]Ekblad A, Wallander H, Godbold DL, et al. The production and turnover of extramatrical mycelium of ectomycorrhizal fungi in forest soils: role in carbon cycling. Plant and Soil. 2013, 366, 1-27. DOI: 10.1007/s11104-013-1630-3.

[54]Palaniyandi U, Muthuswamy A, Sailas B. Endophytic interactions of Trichoderma harzianum in a tropical perennial rhizo-ecosystem. Research Journal of Biotechnology, 2017, 12(3):22-30.

[55]Ainhoa Martínez-Medina, Antonio R, Albacete A, et al. The interaction with arbuscular mycorrhizal fungi or Trichoderma harzianum alters the shoot hormonal profile in melon plants. Phytochemistry. 2011, 72, 223-229. DOI: 10.1016/j.phytochem.2010.11.008.

[56]Giuseppe C, Youssef R, Elena DM. Co‐inoculation of Glomus intraradices and Trichoderma atroviride acts as a biostimulant to promote growth, yield and nutrient uptake of vegetable crops. Journal of the Science of Food and Agriculture. 2015, 95, 1706- 1715. DOI: 10.1002/jsfa.6875.

[57]Sutarman. Effect of ectomycorrhizal fungi and Trichoderma harzianum on the clove (Syzygiumaromaticum L.) seedlings performances. Journal of Physics Conference Series. 2019, 1232, 012022. DOI: 10.1088/1742-6596/1232/1/012022.

[58]Badda N, Yadav K, Kadian N, et al. Impact of arbuscular mycorrhizal fungi with Trichoderma viride and Pseudomonas fluorescens on growth enhancement of genetically modified Bt cotton (Bacillus thuringiensis). Journal of Natural Fibers. 2013, 10, 309-325. DOI: 10.1080/15440478.2013.791913.

[59]Kennedy A, Smith K. Soil microbial diversity and the sustainability of agricultural soils. Plant Soil. 1995, 170:75-86. DOI: 10.1007/BF02183056.

[60]Zhang FG, Huo YQ, Xu XX, et al. Trichoderma improves the growth of Leymus chinensis. Biology & Fertility of Soils. 2018, 54, 685-696. DOI: 10.1007/s00374-018-1292-7.

[61]Gary EH, Charles RH, Viterbo A, et al. Trichoderma species-opportunistic, avirulent plant symbionts. Nature Reviews Microbiology. 2004, 2, 43-56. DOI: 10.1038/nrmicro797.

[62]Hossain MM, Sultana F, Islam S. Plant growthpromoting fungi (PGPF): Phytostimulation and induced systemic resistance. Plant-Microbe Interactions in Agro-Ecological Perspectives. 2017, 135-191. DOI: 10.1007/978-981-10-6593-4_6.

[63]Summerbell R. The inhibitory effect of Trichoderma species and other soil microfungi on formation of mycorrhiza by Laccaria bicolor in vitro. New Phytologist. 2010, 105, 437-448. DOI: 10.1111/j.1469-8137.1987.tb00881.x.

[64]Verena S, Song LF, Erika LQ, et al. Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of a fungal prey. BMC Genomics. 2009, 10, 567. DOI: 10.1186/1471-2164-10-567.

[65]Jong JCD, Mccormack Barbara J, Smirnoff Nicholas, et al. Glycerol generates turgor in rice blast. Nature. 1997, 389, 244. DOI: 10.1038/38418.

[66]Szczepaniak Zuzanna, Cyplik Pawe, Juzwa Wojciech, et al. Antibacterial effect of the Trichoderma viride fungi on soil microbiome during PAH’s biodegradation. International Biodeterioration & Biodegradation. 2015, 104, 170-177. DOI: 10.1016/j.ibiod.2015.06.002.

[67]Jangir M, Sharma S, Sharma S. Non-target effects of Trichoderma on plants and soil microbial communities. Plant Microbe Interface. 2019, 239- 251.DOI: 10.1007/978-3-030-19831-2_10.

[68]Theresa AB, Greg JB. A review of the non-target effects of fungi used to biologically control plant diseases. Agriculture Ecosystems & Environment. 2003, 100:3-16. DOI: 10.1016/s0167-8809(03)00200-7.

[69]Palaniyandi Umadevi, Muthuswamy Anandaraj, Vivek Srivastav, et al. Trichoderma harzianum MTCC 5179 impacts the population and functional dynamics of microbial community in the rhizosphere of black pepper (Piper nigrum L.). Brazilian Journal of Microbiology. 2017, 49(3). DOI: 10.1016/j.bjm.2017.05.011.

[70]Kiers ET, Duhamel M, Beesetty Y, et al. Reciprocal rewards stabilize cooperation in the mycorrhizal symbiosis. Science. 2011, 333(6044): 880-882. DOI: 10.1126/science.1208473.

[71]Oh SY, Park MS, Lim YW. The influence of microfungi on the mycelial growth of ectomycorrhizal fungus Tricholoma matsutake. Microorganisms. 2019, 7(6), 169. DOI: 10.3390/microorganisms7060169.

[72]Wyatt GAK, Kiers ET, Gardner A, et al. A biological market analysis of the plant-mycorrhizal symbiosis. Evolution. 2014, 68 (9), 2603-2618. DOI: 10.1111/evo.12466.

[73]Summerbell RC. From Lamarckian fertilizers to fungal castles: recapturing the pre-1985 literature on endophytic and saprotrophic fungi associated with ectomycorrhizal root systems. Studies in Mycology. 2005, 53:191-256. DOI: 10.3114/sim.53.1.191.

[74]Whipps JM. Prospects and limitations for mycorrhizas in biocontrol of root pathogens. Canadian Journal of Botany. 2011, 82, 1198-1227. DOI: 10.1139/b04-082.

[75]Singh SP, Gaur R. Evaluation of antagonistic and plant growth promoting activities of chitinolytic endophytic actinomycetes associated with medicinal plants against Sclerotium rolfsii in chickpea. Journal of Applied Microbiology. 2016, 121: 506-518. DOI: 10.1111/jam.13176.

[76]Frank DA, Groffman PM. Plant rhizospheric N processes: what we don’t know and why we should care. Ecology. 2009, 90, 1512-1519. DOI: 10.2307/25592653.

[77]Kamran S, Shahid I, Baig DN, et al. Contribution of zinc solubilizing bacteria in growth promotion and zinc content of wheat. Frontiers in Microbiology. 2017, 8, 2593. DOI: 10.3389/fmicb.2017.02593.

[78]Hodge A, Storer K. Arbuscular mycorrhiza and nitrogen: implications for individual plants through to ecosystems. Plant and Soil. 2015, 386: 1-19. DOI: 10.1007/s11104-014-2162-1.

[79]Pritsch K, Garbaye J. Enzyme secretion by ECM fungi and exploitation of mineral nutrients from soil organic matter. Annals of Forest Science. 2011, 68, 25-32. DOI: 10.1007/s13595-010-0004-8.

[80]Louche J, Ali MA, Cloutier-Hurteau B, et al. Efficiency of acid phosphatases secreted from the ectomycorrhizal fungus Hebeloma cylindrosporum to hydrolyse organic phosphorus in podzols. FEMS microbiology ecology. 2010, 73, 323-335. DOI: 10.1111/j.1574-6941.2010.00899.x.

[81]Wurzburger N, Hendrick RL. Rhododendron thickets alter N cycling and soil extracellular enzyme activities in southern Appalachian hardwood forests. Pedobiologia. 2007, 50, 563-576. DOI: 10.1016/j.pedobi.2006.10.001.

[82]Lucas RW, Casper BB. Ectomycorrhizal community and extracellular enzyme activity following simulated atmospheric N deposition. Soil Biology and Biochemistry. 2008, 40, 1662-1669. DOI: 10.1016/j.soilbio.2008.01.025.

[83]De Santiago A, Quintero JM, Avilés M, et al. Effect of Trichoderma asperellum strain T34 on iron nutrition in white lupin. Soil Biology and Biochemistry. 2009, 41: 2453-2459. DOI: 10.1007/s11104-010-0670-1.

[84]Altomare C, Norvell WA, Björkman T, et al. Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22. Applied Environmental Microbiology. 1999, 65, 2926-2933. DOI: 10.1007/s11837-003-0030-1.

[85]A h a n g a r M A , D a r G H , B h a t Z A . G r o w t h response and nutrient uptake of blue pine (Pinus wallichiana) seedlings inoculated with rhizosphere microorganisms under temperate nursery conditions. Annals of Forest Research. 2012, 55, 217-227. DOI: 10.4996/fireecology.0803104.

[86]Colla G, Rouphael Y, Bonini P, et al. Coating seeds with endophytic fungi enhances growth, nutrient uptake, yield and grain quality of winter wheat. International Journal of Plant Production. 2015, 9, 171-189.



DOI: https://doi.org/10.30564/re.v3i2.3286

Refbacks

  • There are currently no refbacks.
Copyright © 2021 Author(s) Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.