ARTICLE IN PRESS MYCRES554_proof 11 March 2009 1/10 mycological research xxx (2009) 1–10 1 58 2 59 3 60 4 61 5 62 6 journal homepage: www.elsevier.com/locate/mycres 63 7 64 8 65 9 Endophytic hyphal compartmentalization is required for 66 10 67 11 68 Q1 successful symbiotic Ascomycota association with root cells 12 69 13 70 a,c c b a, 14 Lobna ABDELLATIF , Sadok BOUZID , Susan KAMINSKYJ , Vladimir VUJANOVIC * 71 15 72 aDepartment of Food and Bioproduct Sciences, University of Saskatchewan, 51 College Drive, Saskatoon, SK S7N 5A8, Canada 16 b 73 Q2 Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK S7N 5E2, Canada 17 cTunis El Manar University, Tunis 1060, Tunisia 74 18 75 19 76 20 article info abstract 77 21 78 22 Article history: Root endophytic fungi are seen as promising alternatives to replace chemical fertilizers 79 23 Received 8 January 2009 and pesticides in sustainable and organicPROOF agriculture systems. Fungal endophytes struc- 80 24 Accepted 24 February 2009 ture formations play key roles in symbiotic intracellular association with plant-roots. To 81 25 Corresponding Editor: John Dighton compare the morphologies of Ascomycete endophytic fungi in wheat, we analyzed growth 82 26 morphologies during endophytic development of hyphae within the cortex of living vs. 83 27 Keywords: dead root cells. Confocal laser scanning microscopy (CLSM) was used to characterize fungal 84 28 Ascomycota cell morphology within lactofuchsin-stained roots. Cell form regularity Ireg and cell growth 85 29 Cell compartmentalization direction Idir, indexes were used to quantify changes in fungal morphology. Endophyte 86 30 Cell morphology fungi in living roots had a variable Ireg and Idir values, low colonization abundance and 87 g 31 Fungal endophytes patchy colonization patterns, whereas the same endophyte species in dead ( -irradiated) 88 roots had consistent form of cells and mostly grew parallel to the root axis. Knot, coil 32 Root 89 Symbiosis and vesicle structures dominated in living roots, as putative symbiotic functional organs. 33 90 Triticum turgidum Finally, an increased hypha septation in living roots might indicate local specialization 34 91 within endophytic Ascomycota. Our results suggested that the applied method could be ex- 35 panded to other septate fungal symbionts (e.g. Basidiomycota). The latter is discussed in 92 36 light of our results and other recent discoveries. 93 37 ª 2009 Published by Elsevier Ltd on behalf of The British Mycological Society. 94 38 95 39 96 40 97 41 98 42 Introduction mycoheterotrophy (Vujanovic & Vujanovic 2007) with en- 99 43 hanced efficiency to control many root diseases (Narisawa 100 44 Fungal endophytes are eukaryotic microorganisms colonizing et al. 1998; Waller et al. 2005; St-Arnaud & Vujanovic 2007). Re- 101 45 healthy tissues of living plants without causing disease symp- cently, enhancement of stress tolerance and disease resis- 102 46 toms (Wilson 1995). They have frequently been reported asso- tance by Piriformospora indica (Basidiomycota) was reported 103 47 ciated with roots, where they appear to protect plants exposed in barley plants (Waller et al. 2005; Deshmukh et al. 2006). How- 104 48 to various abiotic (Marquez et al. 2007; Rodriguez et al. 2008) ever, aside from Deshmukh et al. (2006) who describe the 105 49 and biotic stresses (Narisawa et al.UNCORRECTED2004; Omacini et al. 2001; endorhizal structures produced by P. indica, little data exist 106 50 Rai et al. 2004) and to promote plant growth (Deshmukh & that describe ascomycote endophytic structures in colonized 107 51 Kogel 2007). More specifically, root-colonizing fungi have root of domesticated cereals. Waller et al. (2005) and Desh- 108 et al. 52 been found in mutualistic associations with the majority of mukh (2006) also suggested the different functional 109 terrestrial plant species providing mycovitality and structures of P. indica, including those associated to the 53 110 54 111 55 * Corresponding author. Tel.: þ1 306 966 5048. 112 56 E-mail address: [email protected] 113 57 0953-7562/$ – see front matter ª 2009 Published by Elsevier Ltd on behalf of The British Mycological Society. 114 doi:10.1016/j.mycres.2009.02.013 Please cite this article in press as: Abdellatif L et al., Endophytic hyphal compartmentalization is required for successful sym- biotic Ascomycota association with root cells, Mycological Research (2009), doi:10.1016/j.mycres.2009.02.013 ARTICLE IN PRESS MYCRES554_proof 11 March 2009 2/10 2 L. Abdellatif et al. 115 reproduction cycle, were consequences of the life stages of roots carefully washed, killed by g-irradiation [9.37 Gy per 172 116 colonized plant organs, that is, they were affected by associa- min, 12 h, modified Natarajan & Kesavan (2005)], then 173 117 tion with living vs. dead tissue. Biological significance of the returned to coculture as before for 4 d. Roots placed on PDA 174 118 root cortex cell death in wheat on proliferation of ascomyce- without fungal partners were used as a control. All treatments 175 119 Q3 tous weakly pathogenic Cochlioholus and avirulent Phialophora were repeated twice with three replicates per treatment. After 176 120 isolates has also been suggested (Addy et al. 2005; Deacon & 7 d, all roots were prepared for microscopic analyses, de- 177 121 Henry 1981; Deacon & Lewis 1982). In either case, establish- scribed below. 178 122 ment of the parasitic or mutualistic interaction is the result Observation under a Zeiss Axioskop 2 light microscope 179 123 of a highly sophisticated cross-talk between the partners with 100 magnification showed that the irradiation dose 180 124 (Scha¨fer et al. 2007). Hadacek & Kraus (2002) speculated that used did not reduce cell-wall thickness or destroy the cell- 181 fungal morphology changes may possibly be related to chem- wall, and there was no leakage of cytoplasm (Yu & Wang 125 182 ical variation specifically in the type of carbohydrates present 2006, 2007). 126 183 in the host cell. Whether root-cell structural changes (volume) 127 184 or carbohydrate changes in dead cells (associated with de- Microscopy 128 composition) are involved in fungal morphogenesis is still 185 129 unclear. Living root segments were fixed in formalin, cut into 2 cm seg- 186 130 In this study, we hypothesized that the mutualistic pres- ments, and stained with lactofuchsin (www.sigmaaldrich.- 187 131 sure, two way fungus plant interactions, may differently af- com) as described by Kaminskyj (2008). Stained roots were 188 132 fect the endophytic structures formed in living roots examined with a Zeiss META 510 confocal laser scanning 189 133 compared to those in dead roots. If so, the same endophyte microscope with 514 nm (argon) excitation and LP585 emis- 190 134 might have different cell morphologies before and after root sion filters. Images were collected using a Plan-Neofluar 191 135 senescence. Similarly, endophyte fungi might adopt different 25 N.A.0.8 DIC multi-immersion objective or a C-Apochro- 192 136 colonization patterns depending on the metabolic activity of mat 63 N.A.1.2 phase-contrast water immersion objective. 193 137 the host plant cell tissue. FluorescencePROOF and transmitted images were collected 194 138 The aim of this study was therefore to compare Ascomy- simultaneously. 195 139 cete endophyte colonization patterns and morphologies in 196 140 living and killed roots. To prevent major changes in killed Cell morphometry 197 141 roots, i.e. cell membrane or volume modifications and ar- 198 142 rangement deficiency, we used low-dose g-irradiation to en- All experiments were performed in wheat roots. Fungal cell 199 143 sure no shifting in root-inactive cell structural forms (Yu & morphometry was described quantitatively to compare colo- 200 144 Wang 2006.). According to Natarajan & Kesavan (2005) and nization pattern between living and dead root cells. All of 201 145 Geras’kin et al. (2007), g-irradiated barley meristematic cells the hyphae in ten 100 mm  100 mm areas in randomly selected 202 146 remain biochemically unchanged, so their influence on en- confocal optical sections were assessed for each type of quan- 203 147 dophytic structural formation changes should be minimal. tification. Each host fungus/cell status combination was re- 204 Here, we describe fungus–root interactions in living and peated twice with five replicates. All values are presented as 148 205 killed roots. the averages Æ standard error. Statistical analyses were per- 149 206 SMCD* – Saskatchewan Microbial Collection and Database, formed using Student’s t-test ( p < 0.05). 150 207 College of Agriculture & Bioresources, University of Saskatch- Two indexes were created to assess the shift in fungal 151 ewan, SK, Canada. strain colonization pattern between living and dead root cells. 208 152 These indexes are: 209 153 210 154 Materials and methods (1) Index of fungal cell regularity (Ireg) was employed to discrim- 211 155 inate a shift in fungal cell form. According to Ainsworth 212 156 Two endophytic Ascomycota mitosporic strains (Kiffer & et al. (1971), cell form can be characterized combining 213 157 Morelet 2000) SMCD 2204 (class Dothideomycetes) and SMCD three-dimensional cell structure (rotation about the cen- 214 158 2213 (class Eurotiomycetes) (sensu Hibbet et al. 2007) isolated tral axis) and cell shape distinguished by a length (L)– 215 159 from root of durum wheat Triticum turgidum L. (Saskatchewan, width (W) aspect ratio. In this study, Ireg (L/W) index 216 Canada) were used in this study. 160 values ranged from 1 to 4 – meaning that a cell of 3 mm Q4 217 161 wide would be an aspect ratio of 4 has adjacent septa of 218 162 Endophyte growth experiments 12 mm apart. Length was measured parallel to the hyphal 219 163 UNCORRECTEDaxis, between adjacent septa. Width was perpendicular 220 164 Seeds were surface-sterilized with 95 % ethanol for 10 s, to length and was the greatest cell diameter between 221 165 rinsed in sterile distilled water (SDW) for 10 s, then submerged each pair of septa.