Environmental and Experimental Botany 76 (2012) 33–38 Contents lists available at SciVerse ScienceDirect Environmental and Experimental Botany journa l h omepa g e: www.elsevier.com/locate/envexpbot Direct effects of Trichoderma harzianum strain T-22 on micropropagated shoots of GiSeLa6 ® (Prunus cerasus × Prunus canescens) rootstock Adriano Sofo ∗ , Giuseppe Tataranni, Cristos Xiloyannis, Bartolomeo Dichio, Antonio Scopa Dipartimento di Scienze dei Sistemi Colturali, Forestali e dell’ Ambiente, Università degli Studi della Basilicata, Via dell’Ateneo Lucano 10, 85100 Potenza, Italy a r t i c l e i n f o Article history: Received 21 December 2010 Received in revised form 19 September 2011 Accepted 12 October 2011 Keywords: Cytokinins Enzyme-linked immunosorbent assay Indole-3-acetic acid Microscopic analysis Root acidification capacity Root growth a b s t r a c t Trichoderma harzianum strain T-22 (T22) has the ability of enhancing root growth and plant development. The aim of this research is to explain the biochemical basis of the direct plant-growth-promoting activ- ity of T22. Seven days after the transfer to root-inducing medium, in vitro-cultured shoots of GiSeLa6 ® (Prunus cerasus × Prunus canescens), an important Prunus rootstock for sweet and sour cherry varieties, were inoculated with T22. Indole-3-acetic acid (IAA), trans-zeatin riboside (t-ZR) and dihydrozeatin ribo- side (DHZR) were analyzed by a competitive enzyme-linked immunosorbent assay. Acidification of the medium by plant, T22, and plant + T22 were assessed by three pH indicators, whereas root morphological changes were observed by light and epifluorescence microscopic analysis. The results showed that after T22-inoculation, IAA in leaves and roots significantly increased by 148 and 122%, respectively, whereas DHZR decreased by 83%. Increases in t-ZR were found only in leaves (88%). The ratios auxin/cytokinins changed from 28.5 to 46.6 in leaves, and from 15.0 to 21.2 in roots of un-inoculated and T22-inoculated plants, respectively. Root activity determined a decline of medium acidity, and this effect was more marked in T22-inoculated plants (up to pH 4). Microscopic analysis revealed changes in root cell wall suberification in the exoderm and endoderm, with an increase in suberized cellular layers from 1 to 2–3, and an enhancement of cell wall epifluorescence. During the acclimatisation phase of nursery processes, all these T22-induced changes constitute an advantage, as inoculated plants could acclimatise better, so increasing plant survival in the absence of pesticides. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Trichoderma harzianum has been successfully used for the bio- logical control of many plant pathogens through chemiotropic mycoparasitic interactions with the target fungal organism (Thangavelu et al., 2004; Sahebani and Hadavi, 2008). Several studies demonstrated that T. harzianum produces and excretes mycolytic cell wall degrading enzymes (Carsolio et al., 1994; Haran et al., 1995; Cohen-Kupiec et al., 1999; Noronha and Ulhoa, 2000; Jensen and Shulz, 2004; Ferreira and Ferreira Filho, 2004; Belén Suárez et al., 2005; Yang et al., 2009). The genes encoding some of these enzymes are highly expressed during mycoparasitism (Carsolio et al., 1994; Haran et al., 1995; Cohen-Kupiec et al., 1999; Belén Suárez et al., 2005; Yang et al., 2009) and their transcription is induced by diffusible factors secreted by pathogenic fungi (Cortes Abbreviations: DHZR, dihydrozeatin riboside; ELISA, enzyme-linked immunosorbent assay; IAA, indole-3-acetic acid; T22, Trichoderma harzianum strain T-22; t-ZR, trans-zeatin riboside. ∗ Corresponding author. Tel.: +39 0971 206228; fax: +39 0971 205378. E-mail address: adriano.sofo@unibas.it (A. Sofo). et al., 1998). Therefore, T. harzianum is considered a source of genes for the control of plants diseases (Lorito et al., 1998). On the other hand, it has been reported that T. harzianum has the ability to directly enhance root growth and plant develop- ment in the absence of pathogens (Harman, 2000), and it has been suggested that this could be due to the production of some uniden- tified growth-regulating factors by the fungus (Windham et al., 1986). All these findings indicate the versatility through which T. harzianum can directly manifest biological control activity. In spite of their theoretical and practical importance, the mechanisms responsible for the growth response due to the direct action of T. harzianum on agronomic plants have not been investigated exten- sively. Our research group recently demonstrated that the application of T. harzianum strain T-22 (T22) during the rooting phase of two Prunus spp. rootstocks, under in vitro conditions with plants and T22 alone, resulted in greater shoot lengths, as well as increased numbers of leaves, roots, and stem diameters (Sofo et al., 2010). The same authors observed that the growth-promoting effects of T22 were particularly marked in the genotype GiSeLa6 ® (Prunus cerasus × Prunus canescens), one of the most important commercial rootstocks used for sweet and sour cherry varieties. Furthermore, T. harzianum strain T-22 is particularly important for agronomic 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.10.006