Selection of redox dye and inoculum conditions for the optimisation of respirometric indices in Verticillium and Trichoderma
More details
Hide details
Institute of Agrophysics, Polish Academy of Sciences, Doświadczalna 4, 20-290 Lublin, Poland
Institute for Biological Systems (IBS), Council of National Research of Italy (CNR), Area della Ricerca di Roma 1, via Salaria Km 29, 300, 00015 Monterotondo (RM), Italy
Final revision date: 2023-08-07
Acceptance date: 2023-08-09
Publication date: 2023-08-28
Corresponding author
Karolina Oszust   

Institute of Agrophysics, Polish Academy of Sciences, Doświadczalna 4, 20-290, Lublin, Poland
Int. Agrophys. 2023, 37(3): 279–292
  • Fungal competition for nutrients is measurable and exploitable in biocontrol.
  • Toxic and nutritional effects of dyes affect the results of metabolic profiling.
  • 1% F and 0.5% D BiologTM dyes are reliable with Verticillium and Trichoderma spp.
Understanding which carbon or nitrogen sources the competing fungal species prefer is pivotal for specific biotechnological applications. However, this is not straightforward, as each strain sometimes behaves differently under the experimental conditions adopted. To analyse the trophic overlap of two species, it is necessary to refine diagnostic techniques and exclude variables that may interfere with the measurements. A protocol for establishing the suitability of chromogenic dyes in the analysis of filamentous fungi with phenotype microarrays is described here. The research goal was to determine the most suitable redox dye indicator and its optimal concentration that reacts quantitatively to the respiratory activity of both Verticillium spp. and Trichoderma spp. isolates in the presence of a nitrogen source. The commercial BiologTM Redox Dye Mixes D, E, and F and also TTC (2,3,5-Triphenyltetrazolium chloride), INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride), and MTT solutions were evaluated in selected concentrations. Both their nutritive and toxic effects were quantified. Results showed that 1% “F” and 0.5% “D” BiologTM dyes were appropriate for the functional evaluation of isolates belonging to the Verticillium and Trichoderma genera. They can be used in BiologTM PM-nitrogen plate respiratory assays for a comprehensive functional characterisation of these organisms.
Acin-Albiac M., Filannino P., Gobbetti M., and Di Cagno R., 2020. Microbial high throughput phenomics: The potential of an irreplaceable omics. Computational and Structural Biotechnology J., 18, 2290-2299.
Adedayo A.A., and Babalola O.O., 2023. Fungi that promote plant growth in the rhizosphere boost crop growth. J. Fungi, 9, 239.
Akram S., Ahmed A., He P., He P., Liu Y., Wu Y., Munir S., and He Y., 2023. Uniting the role of endophytic fungi against plant pathogens and their interaction. J. Fungi, 9, 72.
Band N., Kadmon R., Mandel M., and DeMalach N., 2022. Assessing the roles of nitrogen, biomass, and niche dimensionality as drivers of species loss in grassland communities. Proc. National Academy of Sciences, 119, e2112010119.
Bayineni V.K., 2022. Bioremediation of toxic dyes for zero waste In: Bioremediation of Toxic Dyes for Zero Waste, Chapter 4 (Eds C.M. Hussain, R.K. Kadeppagari). John Wiley and Sons, Inc., New York, USA, pp 47-66.
Berg G., Zachow C., Lottmann J., Götz M., Costa R., and Smalla K., 2005. Impact of plant species and site on rhizosphere-associated fungi antagonistic to Verticillium dahliae Kleb. Appl. Environ. Microbiol., 71(8), 4203-13.
Bhadrecha P., Singh S., and Dwibedi V., 2023. A plant’s major strength in rhizosphere: the plant growth promoting rhizobacteria. Archives Microbiol., 205(5), 165.
Braissant O., Astasov-Frauenhoffer M., Waltimo T., and Bonkat G., 2020. A review of methods to determine viability, vitality, and metabolic rates in microbiology. Frontiers Microbiol., 11, 547458.
Broeckling C.D., Broz A.K., Bergelson J., Manter D.K., and Vivanco J.M., 2008. Root exudates regulate soil fungal community composition and diversity. App. Environ.Microbiol., 74(3), 738-44.
Brotman Y., Kapuganti J.G., and Viterbo A., 2010. Trichoderma. Current Biology, 20, R390-R391.
Canfora L., Abu-Samra N., Tartanus M., Łabanowska B.H., Benedetti A., Pinzari F., and Malusà E., 2017. Co-inoculum of Beauveria brongniartii and B. bassiana shows in vitro different metabolic behaviour in comparison to single inoculums. Scientific Reports, 7, 13102.
Chou H.-Y., Chiang M.W.-L., Lin W.-R., Hsieh S.-Y., Jones E.G., Guo S.-Y., and Pang K.-L., 2022. Metabolic activity on Biolog FF MicroPlate suggests organic substrate decomposition by Aspergillus terreus NTOU4989 isolated from Kueishan Island Hydrothermal Vent Field, Taiwan. Fungal Ecol., 101157.
Contreras-Cornejo H.A., Macías-Rodríguez L., Alfaro-Cuevas R., and López-Bucio J., 2014. 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, 27(6), 503-514.
Cruz-Magalhaes V., Nieto-Jacobom M.F., Rostásm M., Echaide-Aquinom J.F., Esquivel-Naranjom E.U., Stewartm A., Loguerciom L.L., and Mendoza-Mendozam A., 2022. Histidine kinase two-component response regulators Ssk1, Skn7 and Rim15 differentially control growth, developmental and volatile organic compounds emissions as stress responses in Trichoderma atroviride. Current Res. Microbial Sci., 3, 100139.
Daayf F., 2015. Verticillium wilts in crop plants: Pathogen invasion and host defence responses. Can. J/f Plant Pathol., 37, 8-20.
Di Mola I., Ottaiano L., Cozzolino E., Marra R., Vitale S., Pironti A., Fiorentino N., and Mori M., 2023. Yield and quality of processing tomato as improved by biostimulants based on Trichoderma sp. and Ascophyllum nodosum and biodegradable mulching films. Agronomy, 13, 901.
Dutta P., Mahanta M., Singh S.B., Thakuria D., Deb L., Kumari A., Upamanya G.K., Boruah S., Dey U., Mishra A.K., Vanlaltani L., VijayReddy D., Heisnam P., and Pandey A.K., 2023. Molecular interaction between plants and Trichoderma species against soil-borne plant pathogens. Frontiers Plant Sci, 14, 1145715.
Frąc M., Kaczmarek J., and Jędryczka M., 2022. Metabolic capacity differentiates Plenodomus lingam from P. biglobosus Subclade’ brassicae’, the causal agents of phoma leaf spotting and stem canker of oilseed rape (Brassica napus) in agricultural ecosystems. Pathogens, 11(1), 50.
Gao L., and Liu X., 2009. A novel two-stage cultivation method to optimise carbon concentration and carbon-to-nitrogen ratio for sporulation of biocontrol fungi. Folia Microbiologica, 54, 142-146.
Gao L., Sun M.H., Liu X.Z., and Che Y.S., 2007. Effects of carbon concentration and carbon to nitrogen ratio on the growth and sporulation of several biocontrol fungi. Mycological Res., 111, 87-92.
Guzmán-Guzmán P., Kumar A., de los Santos-Villalobos S., Parra-Cota F.I., Orozco-Mosqueda Md.C., Fadiji A.E., Hyder S., Babalola O.O., and Santoyo G., 2023. Trichoderma species: our best fungal allies in the biocontrol of plant diseases – A Review. Plants, 12, 432.
Hu Y., Priya A., Chen C., Liang C., Wang W., Wang Q., Lin, C.S.K., and Qi W., 2023. Recent advances in substrate-enzyme interactions facilitating efficient biodegradation of lignocellulosic biomass: A review. Int. Biodeterioration Biodegradation, 180, 105594.
Joseph R., Lasa M., Zhou Y., and Keyhani N.O., 2021. Unique attributes of the laurel wilt fungal pathogen, Raffaelea lauricola, as revealed by metabolic profiling. Pathogens, 10, 528.
Kaushik S., Alatawi A., Djiwanti S.R., Pande A., Skotti E., and Soni V., 2021. Potential of Extremophiles for Bioremediation. In: Microbial Rejuvenation of Polluted Environment (Eds D.G. Panpatte, Y.K. Jhala). Microorganisms for Sustainability, 25. Springer, Singapore.
Lasinio G.J., Pollice A., Pappalettere L., Vannacci G., and Sarrocco S., 2021. A statistical protocol to describe differences among nutrient utilisation patterns of Fusarium spp. and Trichoderma gamsii. Plant Pathology, 70, 1146-1157.
Li J.T., Zhang Y., Chen H., Sun H., Tian W., Li J., Liu X., Zhou S., Fang C., Li B., and Nie M., 2023. Low soil moisture suppresses the thermal compensatory response of microbial respiration. Global Change Biology, 29(3), 874-889.
Lu J., Li X., Tu K., Guan Y., Fung K.-P., and Liu F., 2019. Verticillin A suppresses HGF-induced migration and invasion via repression of the c-Met/FAK/Src pathway in human gastric and cervical cancer cells. OncoTargets and Therapy, 12, 5823.
Malarczyk D.G., Panek J., and Frąc M., 2020. Triplex Real-Time PCR Approach for the detection of crucial fungal berry pathogens – Botrytis spp., Colletotrichum spp. and Verticillium spp. Int. J. Molecular Sci., 21, 8469.
Manzar N., Kashyap A.S., Goutam R.S., Rajawat M.V.S., Sharma P.K., Sharma S.K., and Singh H.V., 2022. Trichoderma: advent of versatile biocontrol agent, its secrets and insights into mechanism of biocontrol potential. Sustainability, 14, 12786.
Massart D.L., Vandeginste B.G., Buydens L.M., Lewi P.J., Smeyers-Verbeke J., and Jong S.D., 1998. Handbook of chemometrics and qualimetrics Part A. In:J. Chem. Inf. Comput. Sci. (Ed. B. Slutsky). Elsevier Science Inc., Amsterdam, Netherlands, 38(6), 1254-1254.
Maynard D.S., Crowther T.W., and Bradford M.A., 2017. Competitive network determines the direction of the diversity-function relationship. Proc. National Academy of Sci., 114(43), 11464-11469.
Morán-Diez M.E., Carrero-Carrón I., Rubio M.B., Jiménez-Díaz R.M., Monte E., and Hermosa R., 2019. Transcriptomic analysis of Trichoderma atroviride overgrowing plant-wilting Verticillium dahliae reveals the role of a new M14 metallocarboxypeptidase CPA1 in biocontrol. Frontiers Microbiol., 10, 1120.
Nuez L., Durand S., Melelli A., Berrin J.-G., Haon M., Drula E., Beaugrand J., D’arras P., Bourmaud A., and Baley C., 2022. Exploring the impact of Verticillium wilt disease on the mechanical properties of elementary flax (Linum usitatissimum L.) fibres. Industrial Crops and Products, 182, 114900.
Oleszek M., Pecio Ł., Kozachok S., Lachowska-Filipiuk Ż., Oszust K., and Frąc M., 2019. Phytochemicals of apple pomace as prospect bio-fungicide agents against mycotoxigenic fungal species – In vitro experiments. Toxins, 11(6), 361.
Oszust K., Cybulska J., and Frąc M., 2020a. How do Trichoderma genus fungi win a nutritional competition battle against soft fruit pathogens? A report on niche overlap nutritional potentiates. Int. J. Molecular Sci., 21(12), 4235.
Oszust K., Pylak M., and Frąc M., 2020b. Patent application No. P.434148. Patent application No. P.434148 (in Polish). The method of obtaining a biopreparation for the naturalization of the raspberry rhizosphere with antagonistic properties to fungal phytopathogens belonging to the genus Botrytis, Verticillium, Colletotrichum and Phytophthora using the fungus strains of the genus Trichoderma, biopreparation for the naturalization of the rhizosphere of raspberry plants, the method of culturing the fungal strains of the genus Trichoderma applications in biopreparation and the composition of the propagation medium for fungi of the genus Trichoderma.
Oszust K., and Frąc M., 2021. First report on the microbial communities of the wild and planted raspberry rhizosphere – A statement on the taxa, processes and a new indicator of functional diversity. Ecological Indicators, 121, 107117.
Oszust K., Panek J., Pertile G., Siczek A., Oleszek M., and Frąc M., 2018. Metabolic and genetic properties of Petriella setifera precultured on waste. Frontiers Microbiol., 9, 115.
Oszust K., Pylak M., and Frąc M., 2021. Trichoderma-based biopreparation with prebiotics supplementation for the naturalisation of raspberry plant rhizosphere. Int. J. Molecular Sci., 22(12), 6356.
Pertile G., Frąc M., Fornal E., Oszust K., Gryta A., and Yaguchi T., 2020. Molecular and metabolic strategies for postharvest detection of heat-resistant fungus Neosartorya fischeri and its discrimination from Aspergillus fumigatus. Postharvest Biology and Technology, 161, 111082.
Pfordt A., Schiwek S., Karlovsky P., von Tiedemann A., 2020. Trichoderma afroharzianum ear rot – A new disease on smaise in Europe. Frontiers Agronomy, 2, 547758.
Pinzari F., Ceci A., Abu-Samra N., Canfora L., Maggi O., and Persiani A., 2016. Phenotype MicroArray™ system in the study of fungal functional diversity and catabolic versatility. Res. Microbiol., 167, 710-722.
Pinzari F., Maggi O., Lunghini D., Di Lonardo D.P., and Persiani A.M., 2017. A simple method for measuring fungal metabolic quotient and comparing carbon use efficiency of different isolates: Application to Mediterranean leaf litter fungi. Plant Biosystems – An Int. J. Dealing with all Aspects Plant Biol., 151, 371-376.
Poveda J., Eugui D., and Abril-Urias P., 2020. Could Trichoderma be a plant pathogen? Successful root colonisation. In: Trichoderma. Host Pathogen Interactions and Applications. (Eds A. Sharma, P. Sharma). Rhizosphere Biology. Springer, Singapore.
Przystas W., Zablocka-Godlewska E., and Grabinska-Sota E., 2015. Efficacy of fungal decolorisation of a mixture of dyes belonging to different classes. Brazilian J. Microbiol., 46(2), 415-424.
Pylak M., Oszust K., and Frąc M., 2019. Review report on the role of bioproducts, biopreparations, biostimulants and microbial inoculants in organic production of fruit. Reviews in Environ. Science and Bio/Technology, 18, 597-616.
Pylak M., Oszust K., and Frąc M., 2020. Searching for new beneficial bacterial isolates of wild raspberries for biocontrol of phytopathogens-antagonistic properties and functional characterisation. Int. J. Molecular Sci., 21(24), 9361.
Pylak M., Oszust K., and Frąc M., 2021. Optimisation of growing medium and preservation methods for plant beneficial bacteria, and formulating a microbial biopreparation for raspberry naturalisation. Agronomy, 11(12), 2521.
Raaijmakers J.M., Paulitz T.C., Steinberg C., Alabouvette C., and Moënne-Loccoz Y., 2009. The rhizosphere: a playground and battlefield for soil-borne pathogens and beneficial microorganisms. Plant Soil, 321, 341-361.
Reusche M., Thole K., Janz D., Truskina J., Rindfleisch S., Drübert C., Polle A., Lipka V., and Teichmann T., 2012. Verticillium infection triggers VASCULAR-RELATED NAC DOMAIN7-dependent de novo xylem formation and enhances drought tolerance in Arabidopsis. Plant Cells, 24(9), 3823-37.
Robb J., 2007. Verticillium tolerance: resistance, susceptibility, or mutualism? Botany, 85, 903-910.
Sen S.K., Raut S., Bandyopadhyay P., and Raut S., 2016. Fungal decolouration and degradation of azo dyes: A review. Fungal Biology Reviews, 30, 112-133.
Septisetyani E.P., Ningrum R.A., Romadhani Y., Wisnuwardhani P.H., and Santoso A., 2014. Optimisation of sodium dodecyl sulphate as a formazan solvent and comparison of 3-(4,-5-dimethylthiazo-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay with wst-1 assay in mcf-7 cells. Indonesian J. Pharmacy, 25, 245.
Singh L., 2017. Biodegradation of synthetic dyes: a mycoremediation approach for degradation/decolourisation of textile dyes and effluents. J. Appl. Biotechnol. Bioengin., 3(5), 430-435.
Sohrabi R., Paasch B.C., Liber J.A., and He S.Y., 2023. Phyllosphere microbiome. An. Review Plant Biol., 74, 539-568.
Spohn M., 2015. Microbial respiration per unit microbial biomass depends on litter layer carbon-to-nitrogen ratio. Biogeosciences, 12, 817-823,
Touchette D., Altshuler I., Raymond-Bouchard I., Fernández-Martínez M.Á., Bourdagesm L.-J., O’Connor B., Riccom A.J., and Whyte L.G., 2022. Microfluidics microbial activity microassay: an automated in situ microbial metabolic detection system. Astrobiology, 22(2), 158-170.
Tyśkiewicz R., Nowak A., Ozimek E., and Jaroszuk-Ściseł J., 2022. Trichoderma: the current status of its application in agriculture for the biocontrol of fungal phytopathogens and stimulation of plant growth. Int. J. Molecular Sci., 23(4), 2329.
Wallis C.M., 2021. Nutritional niche overlap analysis as a method to identify potential biocontrol fungi against trunk pathogens. Biological Control, 66, 559-571.
Zhang D.D., Dai X.F., Klosterman S.J., Subbarao K.V., and Chen J.Y., 2022. The secretome of Verticillium dahliae in collusion with plant defence responses modulates Verticillium wilt symptoms. Biological Reviews, 97(5), 1810-1822.
Zhang G., Bai J., Zhai Y., Jia J., Zhao Q., Wang W., and Hu X., 2023. Microbial diversity and functions in saline soils: A review from a biogeochemical perspective. J. Advanced Res. (in press).