Abstract
In plant-fungus phenotyping, determining fungal hyphal and plant root lengths by digital image analysis can reduce labour and increase data reproducibility. However, the degree of software sophistication is often prohibitive and manual measuring is still used, despite being very time-consuming. We developed the HyLength tool for measuring the lengths of hyphae and roots in in vivo and in vitro systems. The HyLength was successfully validated against manual measures of roots and fungal hyphae obtained from all systems. Compared with manual methods, the HyLength underestimated Medicago sativa roots in the in vivo system and Rhizophagus irregularis hyphae in the in vitro system by about 12 cm per m and allowed to save about 1 h for a single experimental unit. As regards hyphae of R. irregularis in the in vivo system, the HyLength overestimated the length by about 21 cm per m compared with manual measures, but time saving was up to 20.5 h per single experimental unit. Finally, with hyphae of Aspergillus oryzae, the underestimation was about 8 cm per m with a time saving of about 10 min for a single germinating spore. By benchmarking the HyLength against the AnaMorf plugin of the ImageJ/Fiji, we found that the HyLength performed better for dense fungal hyphae, also strongly reducing the measuring time. The HyLength can allow measuring the length over a whole experimental unit, eliminating the error due to sub-area selection by the user and allowing processing a high number of samples. Therefore, we propose the HyLength as a useful freeware tool for measuring fungal hyphae of dense mycelia.
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References
Allen MF, Kitajima K (2013) In situ high-frequency observations of mycorrhizas. New Phytol 200:222–228. https://doi.org/10.1111/nph.12363
Allen JW, Shachar-Hill Y (2009) Sulfur transfer through an arbuscular mycorrhiza. Plant Physiol 149:549–560. https://doi.org/10.1104/pp.108.129866
Altman DG (1991) Practical statistics for medical research. Chapman and Hall, London and New York
Arsenault JL, Poulcur S, Messier C, Guay R (1995) WinRHlZO™, a root-measuring system with a unique overlap correction method. HortScience 30:906. https://doi.org/10.21273/HORTSCI.30.4.906D
Augé RM (2004) Arbuscular mycorrhizae and soil/plant water relations. Can J Soil Sci 84:373–381. https://doi.org/10.4141/S04-002
Avio L, Pellegrino E, Bonari E, Giovannetti M (2006) Functional diversity of arbuscular mycorrhizal fungal isolates in relation to extraradical mycelial networks. New Phytol 172:347–357. https://doi.org/10.1111/j.1469-8137.2006.01839.x
Bago B, Pfeffer PE, Shachar-Hill Y (2000) Carbon metabolism and transport in arbuscular mycorrhizas. Plant Physiol 124:949–958. https://doi.org/10.1104/pp.124.3.949
Barry DJ, Williams GA (2011) Microscopic characterisation of filamentous microbes: towards fully automated morphological quantification through image analysis. J Microsc-Oxford 244:1–20. https://doi.org/10.1111/j.1365-2818.2011.03506.x
Barry DJ, Chan C, Williams GA (2009) Morphological quantification of filamentous fungal development using membrane immobilization and automatic image analysis. J Ind Microbiol Biot 36:787–800. https://doi.org/10.1007/s10295-009-0552-9
Barry DJ, Williams GA, Chan C (2015) Automated analysis of filamentous microbial morphology with AnaMorf. Biotechnol Prog 31:849–852. https://doi.org/10.1002/btpr.2087
Betegón-Putze I, González A, Sevillano X, Blasco-Escámez D, Caño-Delgado AI (2019) My ROOT: a method and software for the semi-automatic measurement of primary root length in Arabidopsis seedlings. Plant J 98:1145–1156. https://doi.org/10.1111/tpj.14297
Brunk M, Sputh S, Doose S, van de Linde S, Terpitz U (2018) HyphaTracker: an ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi. Sci Rep 8:605. https://doi.org/10.1038/s41598-017-19103-1
Bürkert B, Robson A (1994) 65Zn uptake in subterranean clover (Trifolium subterraneum L.) by three vesicular-arbuscular mycorrhizal fungi in a root-free sandy soil. Soil Biol Biochem 26:1117–1124. https://doi.org/10.1016/0038-0717(94)90133-3
Coccina A, Cavagnaro TR, Pellegrino E, Ercoli L, McLaughlin MJ, Watts-Williams SJ (2019) The mycorrhizal pathway of zinc uptake contributes to zinc accumulation in barley and wheat grain. BMC Plant Biol 19:133. https://doi.org/10.1186/s12870-019-1741-y
Cranenbrouck S, Voets L, Bivort C, Renard L, Strullu DG, Declerck S (2005) Methodologies for in vitro cultivation of arbuscular mycorrhizal fungi with root organs. In: Declerck S, Fortin JA, Strullu D (eds) In vitro culture of mycorrhizas. Springer, Berlin, pp 341–375. https://doi.org/10.1007/3-540-27331-X_18
Declerck S, Strullu DG, Plenchette C (1998) Monoxenic culture of the intraradical forms of Glomus sp. isolated from a tropical ecosystem: a proposed methodology for germplasm collection. Mycologia 90:579–585. https://doi.org/10.1080/00275514.1998.12026946
Dhondt S, Van Haerenborgh D, Van Cauwenbergh C, Merks RM, Philips W, Beemster GT, Inzé D (2012) Quantitative analysis of venation patterns of Arabidopsis leaves by supervised image analysis. Plant J 69:553–563. https://doi.org/10.1111/j.1365-313X.2011.04803.x
Ercoli L, Schüßler A, Arduini I, Pellegrino E (2017) Strong increase of durum wheat iron and zinc content by field-inoculation with arbuscular mycorrhizal fungi at different soil nitrogen availabilities. Plant Soil 419:153–167. https://doi.org/10.1007/s11104-017-3319-5
George E, Marschner H, Jakobsen I (1995) Role of arbuscular mycorrhizal fungi in uptake of phosphorus and nitrogen from soil. Crit Rev Biotechnol 15:257–270. https://doi.org/10.3109/07388559509147412
Giovannetti M, Avio L, Fortuna P, Pellegrino E, Sbrana C, Strani P (2006) At the root of the wood wide web: self recognition and nonself incompatibility in mycorrhizal networks. Plant Signal Behav 1:1–5. https://doi.org/10.4161/psb.1.1.2277
Green DC, Newsam R, Jeffries P, Dodd JC, Vilariño A (1994) Quantification of mycelial development of arbuscular mycorrhizal fungi using image analysis. Mycorrhiza 5:105–113. https://doi.org/10.1007/BF00202341
Himmelbauer ML (2004) Estimating length, average diameter and surface area of roots using two different image analyses systems. Plant Soil 260:111–120. https://doi.org/10.1023/B:PLSO.0000030171.28821.55
Hodge A, Helgason T, Fitter AH (2010) Nutritional ecology of arbuscular mycorrhizal fungi. Fungal Ecol 3:267–273. https://doi.org/10.1016/j.funeco.2010.02.002
Jakobsen I, Abbott LK, Robson AD (1992) External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterraneum L. 1. Spread of hyphae and phosphorus inflow into roots. New Phytol 120:371–380. https://doi.org/10.1111/j.1469-8137.1992.tb01077.x
Koch AM, Kuhn G, Fontanillas P, Fumagalli L, Goudet J, Sanders IR (2004) High genetic variability and low local diversity in a population of arbuscular mycorrhizal fungi. P Natl Acad Sci USA 101:2369–2374. https://doi.org/10.1073/pnas.0306441101
Koide RT, Mosse B (2004) A history of research on arbuscular mycorrhiza. Mycorrhiza 14:145–163. https://doi.org/10.1007/s00572-004-0307-4
Kokkoris V, Miles T, Hart MM (2019) The role of in vitro cultivation on asymbiotic trait variation in a single species of arbuscular mycorrhizal fungus. Fungal Biol-UK 123:307–317. https://doi.org/10.1016/j.funbio.2019.01.005
Lawrence I, Lin K (1989) A concordance correlation coefficient to evaluate reproducibility. Biometrics:255–268. https://doi.org/10.2307/2532051
Lehmann A, Rillig MC (2015) Arbuscular mycorrhizal contribution to copper, manganese and iron nutrient concentrations in crops–a meta-analysis. Soil Biol Biochem 81:147–158. https://doi.org/10.1016/j.soilbio.2014.11.013
Li XL, Marschner H, George E (1991) Acquisition of phosphorus and copper by VA-mycorrhizal hyphae and root-to-shoot transport in white clover. Plant Soil 136:49–57. https://doi.org/10.1007/BF02465219
Liu A, Hamel C, Hamilton RI, Ma BL, Smith DL (2000) Acquisition of Cu, Zn, Mn and Fe by mycorrhizal maize (Zea mays L.) grown in soil at different P and micronutrient levels. Mycorrhiza 9:331–336. https://doi.org/10.1007/s005720050277
Lobet G (2017) Image analysis in plant sciences: publish then perish. Trends Plant Sci 22:559–566. https://doi.org/10.1007/BF02465219
Lobet G, Pagès L, Draye X (2011) A novel image-analysis toolbox enabling quantitative analysis of root system architecture. Plant Physiol 157:29–39. https://doi.org/10.1104/pp.111.179895
Lobet G, Draye X, Périlleux C (2013) An online database for plant image analysis software tools. Plant Methods 9:38. https://doi.org/10.1186/1746-4811-9-38
Mathworks Inc (2019) MATLAB version 9.7.0.1165820 (R2019b). Natick, Massachusetts
Meijering E, Jacob M, Sarria JC, Steiner P, Hirling H, Unser M (2004) Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytom Part A 58:167–176. https://doi.org/10.1002/cyto.a.20022
Miransari M, Bahrami HA, Rejali F, Malakouti MJ (2009) Effects of soil compaction and arbuscular mycorrhiza on corn (Zea mays L.) nutrient uptake. Soil Till Res 103:282–290. https://doi.org/10.1016/j.still.2008.10.015
Naeem A, French AP, Wells DM, Pridmore TP (2011) High-throughput feature counting and measurement of roots. Bioinformatics 27:1337–1338. https://doi.org/10.1093/bioinformatics/btr126
Newman EI (1966) A method of estimating the total length of root in a sample. J Appl Ecol:139–145. https://doi.org/10.2307/2401670
Pellegrino E, Ramasamy CK, Sbrana C, Barberi P, Giovannetti M (2010) Selection of infective arbuscular mycorrhizal fungal isolates for field inoculation. Ital J Agron:225–232. https://doi.org/10.4081/ija.2010.225
Pierret A, Gonkhamdee S, Jourdan C, Maeght JL (2013) IJ_Rhizo: an open-source software to measure scanned images of root samples. Plant Soil 373:531–539. https://doi.org/10.1007/s11104-013-1795-9
Qi X, Qi J, Wu Y (2007) RootLM: a simple color image analysis program for length measurement of primary roots in Arabidopsis. Plant Root 1:10–16. https://doi.org/10.3117/plantroot.1.10
Rose L, Lobet G (2019) Accuracy of image analysis tools for functional root traits: a comment on Delory et al. (2017). Methods Ecol Evol 10:702–711. https://doi.org/10.1111/2041-210X.13156
Rovai AP, Baker JD, Ponton MK (2014) Social science research design and statistics: a practitioner’s guide to research methods and IBM SPSS. Watertree Press LLC, Chesapeake
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. https://doi.org/10.1038/nmeth.2019
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. https://doi.org/10.1038/nmeth.2089
Schüßler A, Walker C (2010) The Glomeromycota: a species list with new families and new genera. The Royal Botanic Garden Edinburgh, The Royal Botanic Garden Kew, Botanische Staatssammlung Munich, and Oregon State University
Shen Q, Kirschbaum MU, Hedley MJ, Arbestain MC (2016) Testing an alternative method for estimating the length of fungal hyphae using photomicrography and image processing. PLoS One 11:e0157017. https://doi.org/10.1371/journal.pone.0157017
Smith SE, Read DJ (2008) Mycorrhizal symbiosis. Academic Press, Cambridge
Smith SE, Smith FA (2012) Fresh perspectives on the roles of arbuscular mycorrhizal fungi in plant nutrition and growth. Mycologia 104:1–13. https://doi.org/10.3852/11-229
Smith SE, Gianinazzi-Pearson V, Koide R, Cairney JWG (1994) Nutrient transport in mycorrhizas: structure, physiology and consequences for efficiency of the symbiosis. Plant Soil 159:103–113. https://doi.org/10.1007/BF00000099
St-Arnaud M, Hamel C, Vimard B, Caron M, Fortin JA (1996) Enhanced hyphal growth and spore production of the arbuscular mycorrhizal fungus Glomus intraradices in an in vitro system in the absence of host roots. Mycol Res 100:328–332. https://doi.org/10.1016/S0953-7562(96)80164-X
Tibbett M (2000) Roots, foraging and the exploitation of soil nutrient patches: the role of mycorrhizal symbiosis. Funct Ecol 14:397–399. https://doi.org/10.1046/j.1365-2435.2000.00417.x
van Vuuren MM, Robinson D, Fitter AH, Chasalow SD, Williamson L, Raven JA (1997) Effects of elevated atmospheric CO2 and soil water availability on root biomass, root length, and N, P and K uptake by wheat. New Phytol 135:455–465. https://doi.org/10.1046/j.1469-8137.1997.00682.x
Voets L, Dupré de Boulois H, Renard L, Strullu DG, Declerck S (2005) Development of an autotrophic culture system for the in vitro mycorrhization of potato plantlets. FEMS Microbiol Lett 248:111–118. https://doi.org/10.1016/j.femsle.2005.05.025
Warnock DD, Mummey DL, McBride B, Major J, Lehmann J, Rillig MC (2010) Influences of non-herbaceous biochar on arbuscular mycorrhizal fungal abundances in roots and soils: results from growth-chamber and field experiments. Appl Soil Ecol 46:450–456. https://doi.org/10.1016/j.apsoil.2010.09.002
Zhang H, Wei S, Hu W, Xiao L, Tang M (2017) Arbuscular mycorrhizal fungus Rhizophagus irregularis increased potassium content and expression of genes encoding potassium channels in Lycium barbarum. Front Plant Sci 8:440. https://doi.org/10.3389/fpls.2017.00440
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We acknowledge the Italian Institute of Technology (IIT) for funding the PhD Fellowship of AC under the PhD programme in Agrobiosciences at the Scuola Superiore Sant’Anna of Pisa, Italy.
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AC, EP and LE planned and designed the research; AC performed the research; EDD designed the software tool; AC collected the data; AC, EP and LE analysed and interpreted the data; and AC, EP, EDD, HG, BM and LE wrote the manuscript.
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Cardini, A., Pellegrino, E., Del Dottore, E. et al. HyLength: a semi-automated digital image analysis tool for measuring the length of roots and fungal hyphae of dense mycelia. Mycorrhiza 30, 229–242 (2020). https://doi.org/10.1007/s00572-020-00956-w
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DOI: https://doi.org/10.1007/s00572-020-00956-w