Single or dual inoculation of arbuscular mycorrhizal fungi and rhizobia regulates plant growth and nitrogen acquisition in white clover M., Zou Y., Wu Q., Zhang Z., Kuča K. (2020): Single or dual inoculation of arbuscular mycorrhizal fungi and rhizobia regulates plant growth and nitrogen acquisition in white clover. Plant Soil Environ., 66: 287-294.
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The present work aimed to analyse whether and how single or dual inoculation with arbuscular mycorrhizal fungi (Funneliformis mosseae, Paraglomus occultum, and Rhizophagus intraradices) and rhizobia (Rhizobium trifolii) improved plant growth and stimulated nitrogen (N) acquisition of white clover. AMF inoculation significantly (P < 0.05) increased root nodule number by 117‒173%, and additional Rh considerably stimulated mycorrhizal growth. Single AMF or Rh treatment dramatically increased shoot by 36‒281% and root biomass by 16‒36% than non-inoculated control, and dual inoculation of Rh and P. occultum or R. intraradices further magnified the positive effect. Leaf and root N content, root total soluble protein content, root nitrogenase activity, and amino acid (e.g., alanine, arginine, asparagine, aspartate, phenylalanine, proline, and tryptophan) concentrations were significantly increased by single or dual inoculation, while dual inoculation of AMF and Rh had significantly superior roles than single corresponding AMF or Rh inoculation. These results suggested that AMF and Rh represented synergetic effects on accelerating N acquisition of white clover to some extent, while the combination of P. occultum and Rh was the best.


Abd-Alla M.H., El-Enany A.-W.E., Nafady N.A., Khalaf D.M., Morsy F.M. (2014): Synergistic interaction of Rhizobium leguminosarum bv. viciae and arbuscular mycorrhizal fungi as a plant growth promoting biofertilizers for faba bean (Vicia faba L.) in alkaline soil. Microbiology Research, 169: 49‒58.
Abdel-Fattah G.M., Mohamedin A.H. (2000): Interactions between a vesicular-arbuscular mycorrhizal fungus (Glomus intraradices) and Streptomyces coelicolor and their effects on sorghum plants grown in soil amended with chitin of brawn scales. Biology and Fertility of Soils, 32: 401‒409.
Aliasgharzad N., Neyshabouri M.R., Salimi G. (2006): Effects of arbuscular mycorrhizal fungi and Bradyrhizobium japonicum on drought stress of soybean. Biologia, 61: S324‒S328.
Bethlenfalvay G.J., Ames R.N. (1987): Comparison of two methods for quantifying extraradical mycelium of vesicular-arbuscular mycorrhizal fungi. Soil Science Society of America Journal, 51: 834‒837.
Bever J.D., Dickie I.A., Facelli E., Facelli J.M., Klironomos J., Moora M., Rilling M.C., Stock W.D., Tibbett M., Zobel M. (2010): Rooting theories of plant community ecology in microbial interactions. Trends in Ecology and Evolution, 25: 468‒478.
Bradford M.M. (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248‒254.
Cheng Z., McConkey B.J., Glick B.R. (2010): Proteomic studies of plant-bacterial interactions. Soil Biology and Biochemistry, 42: 1673‒1684.
Cruz C., Egsgaard H., Trujillo C., Ambus P., Requena N., Martins-Loucao M.A., Jakobsen I. (2007): Enzymatic evidence for the key role of arginine in nitrogen translocation by arbuscular mycorrhizal fungi. Plant Physiology, 144: 782‒792.
De Oliveira Júnior J.Q., Jesus E.C., Lisboa F.J., Berbara R.L.L., De Faria S.M. (2017): Nitrogen-fixing bacteria and arbuscular mycorrhizal fungi in Piptadenia gonoacantha (Mart.) Macbr. Brazilian Journal of Microbiology, 48: 95‒100.
Hack C.M., Porta M., Schäufele R., Grimoldi A.A. (2019): Arbuscular mycorrhiza mediated effects on growth, mineral nutrition and biological nitrogen fixation of Melilotus alba Med. in a subtropical grassland soil. Applied Soil Ecology, 134: 38‒44.
He J.D., Chi G.G., Zou Y.N., Shu B., Wu Q.S., Srivastava A.K., Kuča K. (2020): Contribution of glomalin-related soil proteins to soil organic carbon in trifoliate orange. Applied Soil Ecology, 154: 103592.
He J.D., Dong T., Wu H.H., Zou Y.N., Wu Q.S., Kuča K. (2019): Mycorrhizas induce diverse responses of root TIP aquaporin gene expression to drought stress in trifoliate orange. Scientia Horticulturae, 243: 64‒69.
Herridge D.F., Peoples M.B., Boddey R.M. (2008): Global inputs of biological nitrogen fixation in agricultural systems. Plant and Soil, 311: 1‒18.
Hodge A. (2014): Interactions between arbuscular mycorrhizal fungi and organic material substrates. Advances in Applied Microbiology, 89: 47‒99.
Ibiang Y.B., Mitsumoto H., Sakamoto K. (2017): Bradyrhizobia and arbuscular mycorrhizal fungi modulate manganese, iron, phosphorus, and polyphenols in soybean (Glycine max (L.) Merr.) under excess zinc. Environmental and Experimental Botany, 137: 1‒13.
Jin H.R., Jiang D.H., Zhang P.H. (2011): Effect of carbon and nitrogen availability on the metabolism of amino acids in the germinating spores of arbuscular mycorrhizal fungi. Pedosphere, 21: 432‒442.
Larimer A.L., Bever J.D., Clay K. (2010): The interactive effects of plant microbial symbionts: a review and meta-analysis. Symbiosis, 51: 139‒148.
Ledgard S.F., Sprosen M.S., Penno J.W., Rajendram G.S. (2001): Nitrogen fixation by white clover in pastures grazed by dairy cows: temporal variation and effects of nitrogen fertilization. Plant and Soil, 229: 177‒187.
Liyanaarachchi G.V.V., Mahanama K.R.R., Somasiri H.P.P.S., Pumyasiri P.A.N. (2018): Development and validation of a method for direct, underivatized analysis of free amino acids in rice using liquid chromatography-tandem mass spectrometry. Journal of Chromatography A, 1568: 131‒139.
Maillet F., Poinsot V., André O., Puech-Pages V., Haouy A., Gueunier M., Cromer L., Giraudet D., Formey D., Niebel A., Martinez E.A., Driguez H., Bécard G., Dénarié J. (2011): Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature, 469: 58‒63.
Masson-Boivin C., Sachs J.L. (2018): Symbiotic nitrogen fixation by rhizobia – the roots of a success story. Current Opinion in Plant Biology, 44: 7‒15.
Máthá I., Tóth E., Mentes A., Szabó A., Márialigeti K., Schumann P., Felföldi T. (2018): A new Rhizobium species isolated from the water of a crater lake, description of Rhizobium aquaticum sp. nov. Antonie Van Leeuwenhoek, 111: 2175‒2183.
Matsubara Y.I., Okada T., Liu J. (2014): Suppression of fusarium crown rot and increase in several free amino acids in mycorrhizal asparagus. American Journal of Plant Sciences, 5: 235‒240.
Meng L.L., He J.D., Zou Y.N., Wu Q.S., Kuča K. (2020): Mycorrhiza-released glomalin-related soil protein fractions contribute to soil total nitrogen in trifoliate orange. Plant, Soil and Environment, 66: 183‒189.
Musyoka D.M., Njeru E.M., Nyamwange M.M., Maingi J.M. (2020): Arbuscular mycorrhizal fungi and Bradyrhizobium co-inoculation enhances nitrogen fixation and growth of green grams (Vigna radiata L.) under water stress. Journal of Plant Nutrition, 43: 1036‒1047.
Oruru M.B., Njeru E.M. (2016): Upscaling arbuscular mycorrhizal symbiosis and related agroecosystems services in smallholder farming systems. BioMed Research International, 2016: 4376240.
Phillips J.M., Hayman D.S. (1970): Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society, 55: 158‒161.
Shockley F.W., McGraw R.L., Garrett H.E. (2004): Growth and nutrient concentration of two native forage legumes inoculated with Rhizobium and mycorrhiza in Missouri, USA. Agroforestry Systems, 60: 137‒142.
Sood S.G. (2003): Chemotactic response of plant-growth-promoting bacteria towards roots of vesicular-arbuscular mycorrhizal tomato plants. FEMS Microbiology Ecology, 45: 219‒227.
Wu Q.S., He J.D., Srivastava A.K., Zhang F., Zou Y.N. (2019a): Development of propagation technique of indigenous AMF and their inoculation response in citrus. Indian Journal of Agricultural Sciences, 89: 1190‒1194.
Wu Q.S., He J.D., Srivastava A.K., Zou Y.N., Kuča K. (2019b): Mycorrhizas enhance drought tolerance of citrus by altering root fatty acid compositions and their saturation levels. Tree Physiology, 39: 1149‒1158.
Zhang S.Q. (2009): Study on the distribution and number change of rhizobium on alfalfa plants and seeds. [Master’s Thesis]. Lanzhou, Gansu Agricultural University, 51.
Zhang F., Zou Y.N., Wu Q.S., Kuča K. (2020): Arbuscular mycorrhizas modulate root polyamine metabolism to enhance drought tolerance of trifoliate orange. Environmental and Experimental Botany, 171: 103962.
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