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RESEARCH ARTICLE

Yield, zinc efficiencies and biofortification of wheat with zinc sulfate application in soil and foliar nanozinc fertilisation

Arshad Jalal A , Fernando Shintate Galindo B , Leandro Alves Freitas A , Carlos Eduardo da Silva Oliveira A , Bruno Horschut de Lima A , Íngrid Torres Pereira A , Graziela Franceschini Ferraz A , Jeferson Silva de Souza A , Kaway Nunes da Costa A , Thiago Assis Rodrigues Nogueira https://orcid.org/0000-0002-1783-3311 A and Marcelo Carvalho Minhoto Teixeira Filho https://orcid.org/0000-0003-2303-3465 A *
+ Author Affiliations
- Author Affiliations

A Department of Plant Protection, Rural Engineering and Soils (DEFERS), São Paulo State University (UNESP), Postal Code 15385-000, Ilha Solteira, SP, Brazil.

B Center for Nuclear Energy in Agriculture (CENA), University of São Paulo (USP), Postal Code 13416-000, Piracicaba, SP, Brazil.

* Correspondence to: mcm.teixeira-filho@unesp.br

Handling Editor: Shahid Hussain

Crop & Pasture Science - https://doi.org/10.1071/CP21458
Submitted: 28 June 2021  Accepted: 2 December 2021   Published online: 25 May 2022

© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing

Abstract

Context: Agronomic biofortification of wheat (Triticum aestivum L.) with zinc (Zn) is an effective approach to increase grain Zn concentration and productivity and alleviate Zn malnutrition in humans. Foliar Zn application is an alternative strategy to endorse soil Zn deficiency with better grain Zn partitioning.

Aims: This study aimed to better understand dose management of soil and foliar Zn application in wheat for biofortification.

Methods: The objectives was to evaluate the effect of foliar applied nano Zn doses (0, 0.75, 1.5, 3 and 6 kg/ha (zinc oxide, ZnO) 50% at tillering and 50% at grain filling in combination) with soil Zn application (0 and 8 kg/ha, as zinc sulfate) on growth, nutrition, Zn use efficiencies, intake and yield biofortification of wheat in 2019 and 2020 under Brazilian savanna.

Key results: Combined foliar and soil Zn application increased shoot and grains Zn concentration and accumulation with greater dry matter (9.8 and 10.6%) and grain yield (9.8 and 11%) of wheat as compared to control in 2019 and 2020 respectively. Zinc use efficiency (ZnUE), Zn utilisation efficiency and applied Zn recovery improved with soil Zn application and 2.5 kg/ha foliar nano Zn, but decreased with further increase in foliar Zn application. Zn sulfate stood out for increasing crop productivity while foliar spray with nano Zn for better grains biofortification of wheat.

Conclusions: Soil Zn application along with 3 kg/ha of foliar nano Zn increased plant and grains Zn concentration and accumulation, dry matter, grain yield, Zn partitioning index and Zn intake in wheat in tropical conditions of Brazil.

Implications: The combined application of soil and foliar Zn in harsh tropical savannah condition could better improve Zn nutrition, crop growth, and productivity with better Zn biofortification and intake of wheat.

Keywords: agronomic biofortification, foliar zinc, grain yield, soil zinc, wheat, zinc concentration, zinc uptake, zinc use efficiency.


References

Afshar RK, Chen C, Zhou S, Etemadi F, He H, Li Z (2020) Agronomic and economic response of bread wheat to foliar zinc application. Agronomy Journal 112, 4045–4056.
Agronomic and economic response of bread wheat to foliar zinc application.Crossref | GoogleScholarGoogle Scholar |

Ahsin M, Hussain S, Rengel Z, Amir M (2020) Zinc status and its requirement by rural adults consuming wheat from control or zinc-treated fields. Environmental Geochemistry and Health 42, 1877–1892.
Zinc status and its requirement by rural adults consuming wheat from control or zinc-treated fields.Crossref | GoogleScholarGoogle Scholar | 31696401PubMed |

Ajiboye B, Cakmak I, Paterson D, de Jonge MD, Howard DL, Stacey SP, Torun AA, Aydin N, McLaughlin MJ (2015) X-ray fluorescence microscopy of zinc localization in wheat grains biofortified through foliar zinc applications at different growth stages under field conditions. Plant and Soil 392, 357–370.
X-ray fluorescence microscopy of zinc localization in wheat grains biofortified through foliar zinc applications at different growth stages under field conditions.Crossref | GoogleScholarGoogle Scholar |

Akram MA, Depar N, Irfan M (2020) Agronomic zinc biofortification of wheat to improve accumulation, bioavailability, productivity and use efficiency. Eurasian Journal of Soil Science 9, 75–84.
Agronomic zinc biofortification of wheat to improve accumulation, bioavailability, productivity and use efficiency.Crossref | GoogleScholarGoogle Scholar |

Alloway B (2008) ‘Zinc in soils and crop nutrition’, 2nd edn. (International Zinc Association: Brussels and International Fertilizer Industry Association: Paris, France)

Amanullah , Inamullah , Alwahibi MS, Elshikh MS, Alkahtani J, Muhammad A, Khalid S, Imran , Ahmad M, Khan N, Ullah S, Ali I (2020) Phosphorus and zinc fertilization improve zinc biofortification in grains and straw of coarse vs. fine rice genotypes. Agronomy 10, 1155
Phosphorus and zinc fertilization improve zinc biofortification in grains and straw of coarse vs. fine rice genotypes.Crossref | GoogleScholarGoogle Scholar |

Andresen E, Peiter E, Küpper H (2018) Trace metal metabolism in plants. Journal of Experimental Botany 69, 909–954.
Trace metal metabolism in plants.Crossref | GoogleScholarGoogle Scholar | 29447378PubMed |

Aziz MZ, Yaseen M, Abbas T, Naveed M, Mustafa A, Hamid Y, Saeed Q, Xu M-G (2019) Foliar application of micronutrients enhances crop stand, yield and the biofortification essential for human health of different wheat cultivars. Journal of Integrative Agriculture 18, 1369–1378.
Foliar application of micronutrients enhances crop stand, yield and the biofortification essential for human health of different wheat cultivars.Crossref | GoogleScholarGoogle Scholar |

Bhatt R, Hossain A, Sharma P (2020) Zinc biofortification as an innovative technology to alleviate the zinc deficiency in human health: a review. Open Agriculture 5, 176–187.
Zinc biofortification as an innovative technology to alleviate the zinc deficiency in human health: a review.Crossref | GoogleScholarGoogle Scholar |

Bollinedi H, Yadav AK, Vinod KK, Krishnan SG, Bhowmick PK, Nagarajan M, Neeraja CN, Ellur RK, Singh AK (2020) Genome-wide association study reveals novel marker-trait associations (MTAs) governing the localization of Fe and Zn in the rice grain. Frontiers in Genetics 11, 1655
Genome-wide association study reveals novel marker-trait associations (MTAs) governing the localization of Fe and Zn in the rice grain.Crossref | GoogleScholarGoogle Scholar |

Boonchuay P, Cakmak I, Rerkasem B, Prom-U-Thai C (2013) Effect of different foliar zinc application at different growth stages on seed zinc concentration and its impact on seedling vigor in rice. Soil Science and Plant Nutrition 59, 180–188.
Effect of different foliar zinc application at different growth stages on seed zinc concentration and its impact on seedling vigor in rice.Crossref | GoogleScholarGoogle Scholar |

Cakmak I (2008) Enrichment of cereal grains with zinc: agronomic or genetic biofortification? Plant and Soil 302, 1–17.
Enrichment of cereal grains with zinc: agronomic or genetic biofortification?Crossref | GoogleScholarGoogle Scholar |

Cakmak I, Kalayci M, Kaya Y, Torun AA, Aydin N, Wang Y, Arisoy Z, Erdem H, Yazici A, Gokmen O, Ozturk L, Horst WJ (2010) Biofortification and localization of zinc in wheat grain. Journal of Agricultural and Food Chemistry 58, 9092–9102.
Biofortification and localization of zinc in wheat grain.Crossref | GoogleScholarGoogle Scholar | 23654236PubMed |

Cantarella H, van Raij B, Camargo CEO (1997) Cereals. In ‘Liming and fertilization recommendations for the State of São Paulo’. Boletim técnico, 100. (Eds B van Raij, H Cantarella, JA Quaggio, AMC Furlani) p. 285. (Instituto Agronômico de Campinas: Campinas) (In Portuguese)

Chattha MU, Hassan MU, Khan I, Chattha MB, Mahmood A, Nawaz M, Subhani MN, Kharal M, Khan S (2017) Biofortification of wheat cultivars to combat zinc deficiency. Frontiers in Plant Science 8, 281
Biofortification of wheat cultivars to combat zinc deficiency.Crossref | GoogleScholarGoogle Scholar | 28352273PubMed |

de Lima Lessa JH, Araujo AM, Ferreira LA, da Silva Júnior EC, de Oliveira C, Corguinha APB, Martins FAD, de Carvalho HWP, Guilherme LRG, Lopes G (2019) Agronomic biofortification of rice (Oryza sativa L.) with selenium and its effect on element distributions in biofortified grains. Plant and Soil 444, 331–342.
Agronomic biofortification of rice (Oryza sativa L.) with selenium and its effect on element distributions in biofortified grains.Crossref | GoogleScholarGoogle Scholar |

Dhaliwal SS, Ram H, Shukla AK, Mavi GS (2019) Zinc biofortification of bread wheat, triticale, and durum wheat cultivars by foliar zinc fertilization. Journal of Plant Nutrition 42, 813–822.
Zinc biofortification of bread wheat, triticale, and durum wheat cultivars by foliar zinc fertilization.Crossref | GoogleScholarGoogle Scholar |

Doolette CL, Read TL, Li C, Scheckel KG, Donner E, Kopittke PM, Schjoerring JK, Lombi E (2018) Foliar application of zinc sulphate and zinc EDTA to wheat leaves: differences in mobility, distribution, and speciation. Journal of Experimental Botany 69, 4469–4481.
Foliar application of zinc sulphate and zinc EDTA to wheat leaves: differences in mobility, distribution, and speciation.Crossref | GoogleScholarGoogle Scholar | 29931117PubMed |

Doolette CL, Read TL, Howell NR, Cresswell T, Lombi E (2020) Zinc from foliar-applied nanoparticle fertiliser is translocated to wheat grain: a 65Zn radiolabelled translocation study comparing conventional and novel foliar fertilisers. Science of The Total Environment 749, 142369
Zinc from foliar-applied nanoparticle fertiliser is translocated to wheat grain: a 65Zn radiolabelled translocation study comparing conventional and novel foliar fertilisers.Crossref | GoogleScholarGoogle Scholar | 33370927PubMed |

dos Santos WJR, Curi N, Silva SHG, de Araújo EF, Marques JJ (2013) Pedotransfer functions for water retention in different soil classes from the center-southern Rio Grande do Sul State. Ciência e Agrotecnologia 37, 49–60.
Pedotransfer functions for water retention in different soil classes from the center-southern Rio Grande do Sul State.Crossref | GoogleScholarGoogle Scholar |

Du W, Yang J, Peng Q, Liang X, Mao H (2019) Comparison study of zinc nanoparticles and zinc sulphate on wheat growth: from toxicity and zinc biofortification. Chemosphere 227, 109–116.
Comparison study of zinc nanoparticles and zinc sulphate on wheat growth: from toxicity and zinc biofortification.Crossref | GoogleScholarGoogle Scholar | 30986592PubMed |

Fageria NK, Baligar VC (2001) Improving nutrient use efficiency of annual crops in Brazilian acid soils for sustainable crop production. Communications in Soil Science and Plant Analysis 32, 1303–1319.
Improving nutrient use efficiency of annual crops in Brazilian acid soils for sustainable crop production.Crossref | GoogleScholarGoogle Scholar |

Fageria NK, Baligar VC, Clark RB (2002) Micronutrients in crop production. Advances in Agronomy 77, 185–268.
Micronutrients in crop production.Crossref | GoogleScholarGoogle Scholar |

Fageria NK, dos Santos AB, Cobucci T (2011) Zinc nutrition of lowland rice. Communications in Soil Science and Plant Analysis 42, 1719–1727.
Zinc nutrition of lowland rice.Crossref | GoogleScholarGoogle Scholar |

Fageria NK, Moraes MF, Ferreira EPB, Knupp AM (2012) Biofortification of trace elements in food crops for human health. Communications in Soil Science and Plant Analysis 43, 556–570.
Biofortification of trace elements in food crops for human health.Crossref | GoogleScholarGoogle Scholar |

Fernández V, Brown PH (2013) From plant surface to plant metabolism: the uncertain fate of foliar-applied nutrients. Frontiers in Plant Science 4, 289
From plant surface to plant metabolism: the uncertain fate of foliar-applied nutrients.Crossref | GoogleScholarGoogle Scholar | 23914198PubMed |

Firdous S, Agarwal BK, Chhabra V, Kumar A (2020) Zinc allocation and its re-translocation in wheat at different growth stages. Plant Archives 20, 8653–8659.

Gomez-Coronado F, Poblaciones MJ, Almeida AS, Cakmak I (2016) Zinc (Zn) concentration of bread wheat grown under Mediterranean conditions as affected by genotype and soil/foliar Zn application. Plant and Soil 401, 331–346.
Zinc (Zn) concentration of bread wheat grown under Mediterranean conditions as affected by genotype and soil/foliar Zn application.Crossref | GoogleScholarGoogle Scholar |

Gupta N, Ram H, Kumar B (2016) Mechanism of zinc absorption in plants: uptake, transport, translocation and accumulation. Reviews in Environmental Science and Bio/Technology 15, 89–109.
Mechanism of zinc absorption in plants: uptake, transport, translocation and accumulation.Crossref | GoogleScholarGoogle Scholar |

Hafeez FY, Abaid-Ullah M, Hassan MN (2013) Plant growth-promoting rhizobacteria as zinc mobilizers: a promising approach for cereals biofortification. In ‘Bacteria in agrobiology: crop productivity’. (Eds DK Maheshwari, M Saraf, A Aeron) pp. 217–235. (Springer: Berlin, Heidelberg)

Haider MU, Hussain M, Farooq M (2019) Optimizing zinc seed coating treatments for improving growth, productivity and grain biofortification of mungbean. Soil & Environment 38, 97–102.
Optimizing zinc seed coating treatments for improving growth, productivity and grain biofortification of mungbean.Crossref | GoogleScholarGoogle Scholar |

Haider MU, Hussain M, Farooq M, Nawaz A (2020) Zinc nutrition for improving the productivity and grain biofortification of mungbean. Journal of Soil Science and Plant Nutrition 20, 1321–1335.

Hussain S, Maqsood MA, Aziz T, Basra SMA (2013) Zinc bioavailability response curvature in wheat grains under incremental zinc applications. Archives of Agronomy and Soil Science 59, 1001–1016.
Zinc bioavailability response curvature in wheat grains under incremental zinc applications.Crossref | GoogleScholarGoogle Scholar |

Jalal A, Shah S, Teixeira Filho MCM, Khan A, Shah T, Hussain M, Younis M, Ilyas M (2020a) Yield and phenological indices of wheat as affected by exogenous fertilization of zinc and iron. Brazilian Journal of Agricultural Sciences/Revista Brasileira de Ciências Agrárias 15, e7730
Yield and phenological indices of wheat as affected by exogenous fertilization of zinc and iron.Crossref | GoogleScholarGoogle Scholar |

Jalal A, Shah S, Teixeira Filho MCM, Khan A, Shah T, Ilyas M, Rosa PAL (2020b) Agro-biofortification of zinc and iron in wheat grains. Gesunde Pflanzen 72, 227–236.
Agro-biofortification of zinc and iron in wheat grains.Crossref | GoogleScholarGoogle Scholar |

Jalal A, Galindo FS, Boleta EHM, Oliveira CEdeS, dos Reis AR, Nogueira TAR, Moretti Neto MJ, Mortinho ES, Fernandes GC, Teixeira Filho MCM (2021) Common bean yield and zinc use efficiency in association with diazotrophic bacteria co-inoculations. Agronomy 11, 959
Common bean yield and zinc use efficiency in association with diazotrophic bacteria co-inoculations.Crossref | GoogleScholarGoogle Scholar |

Li M, Yang XW, Tian XH, Wang SX, Chen YL (2014) Effect of nitrogen fertilizer and foliar zinc application at different growth stages on zinc translocation and utilization efficiency in winter wheat. Cereal Research Communication 42, 81–90.
Effect of nitrogen fertilizer and foliar zinc application at different growth stages on zinc translocation and utilization efficiency in winter wheat.Crossref | GoogleScholarGoogle Scholar |

Liu D-Y, Liu Y-M, Zhang W, Chen X-P, Zou C-Q (2019) Zinc uptake, translocation, and remobilization in winter wheat as affected by soil application of Zn fertilizer. Frontiers in Plant Science 10, 426
Zinc uptake, translocation, and remobilization in winter wheat as affected by soil application of Zn fertilizer.Crossref | GoogleScholarGoogle Scholar | 31057568PubMed |

Liu D-Y, Zhang W, Liu Y-M, Chen X-P, Zou C-Q (2020) Soil application of zinc fertilizer increases maize yield by enhancing the kernel number and kernel weight of inferior grains. Frontiers in Plant Science 11, 188
Soil application of zinc fertilizer increases maize yield by enhancing the kernel number and kernel weight of inferior grains.Crossref | GoogleScholarGoogle Scholar | 32180784PubMed |

Malavolta E, Vitti GC, Oliveira SA (1997) ‘Evaluation of the nutritional status of plants: principles and applications’, 2nd edn. p. 319. (Potafos: Piracicaba) (In Portuguese)

Maxfield L, Crane JS (2019) Zinc deficiency. StatPearls [Internet]. StatPearls Publishing. Available at https://www.ncbi.nlm.nih.gov/books/NBK493231/.

Noulas C, Tziouvalekas M, Karyotis T (2018) Zinc in soils, water and food crops. Journal of Trace Elements in Medical and Biology 49, 252–260.
Zinc in soils, water and food crops.Crossref | GoogleScholarGoogle Scholar |

Palacio-Márquez A, Ramírez-Estrada CA, Gutiérrez-Ruelas NJ, Sánchez E, Ojeda-Barrios DL, Chávez-Mendoza C, Sida-Arreola JP (2021) Efficiency of foliar application of zinc oxide nanoparticles versus zinc nitrate complexed with chitosan on nitrogen assimilation, photosynthetic activity, and production of green beans (Phaseolus vulgaris L.). Scientia Horticulturae 288, 110297
Efficiency of foliar application of zinc oxide nanoparticles versus zinc nitrate complexed with chitosan on nitrogen assimilation, photosynthetic activity, and production of green beans (Phaseolus vulgaris L.).Crossref | GoogleScholarGoogle Scholar |

Penn CJ, Camberato JJ (2019) A critical review on soil chemical processes that control how soil pH affects phosphorus availability to plants. Agriculture 9, 120
A critical review on soil chemical processes that control how soil pH affects phosphorus availability to plants.Crossref | GoogleScholarGoogle Scholar |

Poniedziałek B, Perkowska K, Rzymski P (2020) Food fortification: what’s in it for the malnourished world? In ‘Vitamins and minerals biofortification of edible plants’. (Ed. N Benkeblia) pp. 27–44. (John Wiley & Sons Ltd.)
| Crossref |

R Core Team (2015) R: a Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Available at https://www.R-project.org/

Read TL, Doolette CL, Howell NR, Kopittke PM, Cresswell T, Lombi E (2021) Zinc accumulates in the nodes of wheat following the foliar application of 65Zn oxide nano-and microparticles. Environmental Science & Technology 55, 13523–13531.
Zinc accumulates in the nodes of wheat following the foliar application of 65Zn oxide nano-and microparticles.Crossref | GoogleScholarGoogle Scholar |

Rehman R, Asif M, Cakmak I, Ozturk L (2021) Differences in uptake and translocation of foliar-applied Zn in maize and wheat. Plant and Soil 462, 235–244.
Differences in uptake and translocation of foliar-applied Zn in maize and wheat.Crossref | GoogleScholarGoogle Scholar |

Rengel Z, Graham RD (1996) Uptake of zinc from chelate-buffered nutrient solutions by wheat genotypes differing in zinc efficiency. Journal of Experimental Botany 47, 217–226.
Uptake of zinc from chelate-buffered nutrient solutions by wheat genotypes differing in zinc efficiency.Crossref | GoogleScholarGoogle Scholar |

Rossi L, Fedenia LN, Sharifan H, Ma X, Lombardini L (2019) Effects of foliar application of zinc sulfate and zinc nanoparticles in coffee (Coffea arabica L.) plants. Plant Physiology and Biochemistry 135, 160–166.
Effects of foliar application of zinc sulfate and zinc nanoparticles in coffee (Coffea arabica L.) plants.Crossref | GoogleScholarGoogle Scholar | 30553137PubMed |

Singh P, Shukla AK, Behera SK, Tiwari PK (2019) Zinc application enhances superoxide dismutase and carbonic anhydrase activities in zinc-efficient and zinc-inefficient wheat genotypes. Journal of Soil Science and Plant Nutrition 19, 477–487.
Zinc application enhances superoxide dismutase and carbonic anhydrase activities in zinc-efficient and zinc-inefficient wheat genotypes.Crossref | GoogleScholarGoogle Scholar |

Suman A, Shukla L, Marag PS, Verma P, Gond S, Prasad JS (2020) Potential use of plant colonizing Pantoea as generic plant growth promoting bacteria for cereal crops. Journal of Environmental Biology 41, 987–994.
Potential use of plant colonizing Pantoea as generic plant growth promoting bacteria for cereal crops.Crossref | GoogleScholarGoogle Scholar |

Teixeira PC, Donagemma GK, Fontana A, Teixeira WG (2017) ‘Manual of soil analysis methods’, p. 575. (Centro nacional de pesquisa de solos, Embrapa: Rio de Janeiro) (In Portuguese)

United States Department of Agriculture – USDA (2010) ‘Keys to soil taxonomy’, 11th edn. p. 338. (USDA, NRCS: Washington, DC, USA)

USDA, Foreign Agriculture Services; Brazil Grain and Feed Annual (2020) Available at http://www.abitrigo.com.br/wp-content/uploads/2019/09/CONSUMO-MUNDIAL-DE-TRIGO-15_16-20_21.pdf. (Accessed 29 May 2021)

Uwitonze AM, Ojeh N, Murererehe J, Atfi A, Razzaque MS (2020) Zinc adequacy is essential for the maintenance of optimal oral health. Nutrients 12, 949
Zinc adequacy is essential for the maintenance of optimal oral health.Crossref | GoogleScholarGoogle Scholar |

van Raij B, Andrade JC, Cantarella H, Quaggio JA (2001) ‘Chemical analysis for fertility evaluation of tropical soils’, p. 285. (IAC: Campinas) (In Portuguese)

Wang L, Xia H, Li X, Qiao Y, Xue Y, Jiang X, Yan W, Liu Y, Xue Y, Kong L (2021) Source–sink manipulation affects accumulation of zinc and other nutrient elements in wheat grains. Plants 10, 1032
Source–sink manipulation affects accumulation of zinc and other nutrient elements in wheat grains.Crossref | GoogleScholarGoogle Scholar | 34065615PubMed |

Wessels I, Rolles B, Rink L (2020) The potential impact of zinc supplementation on COVID-19 pathogenesis. Frontiers in Immunology 11, 1712
The potential impact of zinc supplementation on COVID-19 pathogenesis.Crossref | GoogleScholarGoogle Scholar | 32754164PubMed |

Yaseen MK, Hussain S (2020) Zinc-biofortified wheat required only a medium rate of soil zinc application to attain the targets of zinc biofortification. Archives of Agronomy and Soil Science 67, 551–562.
Zinc-biofortified wheat required only a medium rate of soil zinc application to attain the targets of zinc biofortification.Crossref | GoogleScholarGoogle Scholar |

Zhao A, Wang B, Tian X, Yang X (2020) Combined soil and foliar ZnSO4 application improves wheat grain Zn concentration and Zn fractions in a calcareous soil. European Journal of Soil Science 71, 681–694.
Combined soil and foliar ZnSO4 application improves wheat grain Zn concentration and Zn fractions in a calcareous soil.Crossref | GoogleScholarGoogle Scholar |

Zou CQ, Zhang YQ, Rashid A, Ram H, Savasli E, Arisoy RZ, Ortiz-Monasterio I, Simunji S, Wang ZH, Sohu V, Hassan M (2012) Biofortification of wheat with zinc through zinc fertilization in seven countries. Plant and Soil 361, 119–130.
Biofortification of wheat with zinc through zinc fertilization in seven countries.Crossref | GoogleScholarGoogle Scholar |