Endophytic hyphal compartmentalization is required for successful ...

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successful symbiotic Ascomycota association with. Q1 root cells ... within endophytic Ascomycota. ..... cellular form transition is also a key feature in the biology of.
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Endophytic hyphal compartmentalization is required for successful symbiotic Ascomycota association with root cells Q1 Lobna ABDELLATIFa,c, Sadok BOUZIDc, Susan KAMINSKYJb, Vladimir VUJANOVICa,* a

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Department of Food and Bioproduct Sciences, University of Saskatchewan, 51 College Drive, Saskatoon, SK S7N 5A8, Canada

b Q2 Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK S7N 5E2, Canada c

Tunis El Manar University, Tunis 1060, Tunisia

abstract

Article history:

Root endophytic fungi are seen as promising alternatives to replace chemical fertilizers

Received 8 January 2009

and pesticides in sustainable and organic agriculture systems. Fungal endophytes struc-

Accepted 24 February 2009

ture formations play key roles in symbiotic intracellular association with plant-roots. To

Corresponding Editor: John Dighton

compare the morphologies of Ascomycete endophytic fungi in wheat, we analyzed growth

Keywords:

dead root cells. Confocal laser scanning microscopy (CLSM) was used to characterize fungal

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article info

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morphologies during endophytic development of hyphae within the cortex of living vs. cell morphology within lactofuchsin-stained roots. Cell form regularity Ireg and cell growth

Cell compartmentalization

direction Idir, indexes were used to quantify changes in fungal morphology. Endophyte

Cell morphology

fungi in living roots had a variable Ireg and Idir values, low colonization abundance and

Fungal endophytes

patchy colonization patterns, whereas the same endophyte species in dead (g-irradiated)

Root

roots had consistent form of cells and mostly grew parallel to the root axis. Knot, coil

Symbiosis

and vesicle structures dominated in living roots, as putative symbiotic functional organs.

Triticum turgidum

Finally, an increased hypha septation in living roots might indicate local specialization

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Ascomycota

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within endophytic Ascomycota. Our results suggested that the applied method could be expanded to other septate fungal symbionts (e.g. Basidiomycota). The latter is discussed in light of our results and other recent discoveries.

Introduction

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ª 2009 Published by Elsevier Ltd on behalf of The British Mycological Society.

Fungal endophytes are eukaryotic microorganisms colonizing healthy tissues of living plants without causing disease symptoms (Wilson 1995). They have frequently been reported associated with roots, where they appear to protect plants exposed to various abiotic (Marquez et al. 2007; Rodriguez et al. 2008) and biotic stresses (Narisawa et al. 2004; Omacini et al. 2001; Rai et al. 2004) and to promote plant growth (Deshmukh & Kogel 2007). More specifically, root-colonizing fungi have been found in mutualistic associations with the majority of terrestrial plant species providing mycovitality and

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mycoheterotrophy (Vujanovic & Vujanovic 2007) with enhanced efficiency to control many root diseases (Narisawa et al. 1998; Waller et al. 2005; St-Arnaud & Vujanovic 2007). Recently, enhancement of stress tolerance and disease resistance by Piriformospora indica (Basidiomycota) was reported in barley plants (Waller et al. 2005; Deshmukh et al. 2006). However, aside from Deshmukh et al. (2006) who describe the endorhizal structures produced by P. indica, little data exist that describe ascomycote endophytic structures in colonized root of domesticated cereals. Waller et al. (2005) and Deshmukh et al. (2006) also suggested the different functional structures of P. indica, including those associated to the

* Corresponding author. Tel.: þ1 306 966 5048. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2009 Published by Elsevier Ltd on behalf of The British Mycological Society. 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 symbiotic Ascomycota association with root cells, Mycological Research (2009), doi:10.1016/j.mycres.2009.02.013

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Microscopy

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roots carefully washed, killed by g-irradiation [9.37 Gy per min, 12 h, modified Natarajan & Kesavan (2005)], then returned to coculture as before for 4 d. Roots placed on PDA without fungal partners were used as a control. All treatments were repeated twice with three replicates per treatment. After 7 d, all roots were prepared for microscopic analyses, described below. Observation under a Zeiss Axioskop 2 light microscope with 100 magnification showed that the irradiation dose used did not reduce cell-wall thickness or destroy the cellwall, and there was no leakage of cytoplasm (Yu & Wang 2006, 2007).

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Living root segments were fixed in formalin, cut into 2 cm segments, and stained with lactofuchsin (www.sigmaaldrich.com) as described by Kaminskyj (2008). Stained roots were examined with a Zeiss META 510 confocal laser scanning microscope with 514 nm (argon) excitation and LP585 emission filters. Images were collected using a Plan-Neofluar 25 N.A.0.8 DIC multi-immersion objective or a C-Apochromat 63 N.A.1.2 phase-contrast water immersion objective. Fluorescence and transmitted images were collected simultaneously.

Cell morphometry

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reproduction cycle, were consequences of the life stages of 115 colonized plant organs, that is, they were affected by associa116 tion with living vs. dead tissue. Biological significance of the 117 root cortex cell death in wheat on proliferation of ascomyce118 119 Q3 tous weakly pathogenic Cochlioholus and avirulent Phialophora isolates has also been suggested (Addy et al. 2005; Deacon & 120 Henry 1981; Deacon & Lewis 1982). In either case, establish121 ment of the parasitic or mutualistic interaction is the result 122 of a highly sophisticated cross-talk between the partners 123 (Scha¨fer et al. 2007). Hadacek & Kraus (2002) speculated that 124 fungal morphology changes may possibly be related to chem125 ical variation specifically in the type of carbohydrates present 126 in the host cell. Whether root-cell structural changes (volume) 127 or carbohydrate changes in dead cells (associated with de128 composition) are involved in fungal morphogenesis is still 129 unclear. 130 In this study, we hypothesized that the mutualistic pres131 sure, two way fungus plant interactions, may differently af132 fect the endophytic structures formed in living roots 133 compared to those in dead roots. If so, the same endophyte 134 might have different cell morphologies before and after root 135 senescence. Similarly, endophyte fungi might adopt different colonization patterns depending on the metabolic activity of 136 the host plant cell tissue. 137 The aim of this study was therefore to compare Ascomy138 cete endophyte colonization patterns and morphologies in 139 living and killed roots. To prevent major changes in killed 140 roots, i.e. cell membrane or volume modifications and ar141 rangement deficiency, we used low-dose g-irradiation to en142 sure no shifting in root-inactive cell structural forms (Yu & 143 Wang 2006.). According to Natarajan & Kesavan (2005) and 144 Geras’kin et al. (2007), g-irradiated barley meristematic cells 145 remain biochemically unchanged, so their influence on en146 dophytic structural formation changes should be minimal. 147 Here, we describe fungus–root interactions in living and 148 killed roots. 149 SMCD* – Saskatchewan Microbial Collection and Database, 150 College of Agriculture & Bioresources, University of Saskatch151 ewan, SK, Canada. 152 153 Materials and methods 154 155 Two endophytic Ascomycota mitosporic strains (Kiffer & 156 Morelet 2000) SMCD 2204 (class Dothideomycetes) and SMCD 157 2213 (class Eurotiomycetes) (sensu Hibbet et al. 2007) isolated 158 from root of durum wheat Triticum turgidum L. (Saskatchewan, 159 Canada) were used in this study. 160 161 Endophyte growth experiments 162 163 Seeds were surface-sterilized with 95 % ethanol for 10 s, 164 rinsed in sterile distilled water (SDW) for 10 s, then submerged 165 for 3 min in 5 % sodium hypochlorite, rinsed 3 times in SDW, 166 and placed on potato dextrose agar (PDA) for germination. 167 Ten seeds were spread over 9 cm Petri plates. After 3 d of incu168 bation (Precision Fisher Scientific Inc., Incubator MDL3EG) at 169 21  C in darkness, half of the young seedlings were co-cul170 tured in association with fungal mycelia (5 mm2) for 4 d. Half 171 of the 3 d old seedlings were removed from the medium, their

All experiments were performed in wheat roots. Fungal cell morphometry was described quantitatively to compare colonization pattern between living and dead root cells. All of the hyphae in ten 100 mm  100 mm areas in randomly selected confocal optical sections were assessed for each type of quantification. Each host fungus/cell status combination was repeated twice with five replicates. All values are presented as the averages  standard error. Statistical analyses were performed using Student’s t-test ( p < 0.05). Two indexes were created to assess the shift in fungal strain colonization pattern between living and dead root cells. These indexes are: (1) Index of fungal cell regularity (Ireg) was employed to discriminate a shift in fungal cell form. According to Ainsworth et al. (1971), cell form can be characterized combining three-dimensional cell structure (rotation about the central axis) and cell shape distinguished by a length (L)– width (W) aspect ratio. In this study, Ireg (L/W) index values ranged from 1 to 4 – meaning that a cell of 3 mm wide would be an aspect ratio of 4 has adjacent septa of 12 mm apart. Length was measured parallel to the hyphal axis, between adjacent septa. Width was perpendicular to length and was the greatest cell diameter between each pair of septa. Within calculated Ireg scale (1–4), two distinct shape groups were distinguished based on variability measured between minimal (Iregmin) and maximal (Iregmax) aspect ratio. Type I with cylindrical or regular cell form was characterized by an Iregmax > 2 (Fig 1b), and Type II with round (globose to subglobose) or irregular cell form was characterized by an Iregmax < 2 (Fig 1a). In both types, no differences were observed in

Please cite this article in press as: Abdellatif L et al., Endophytic hyphal compartmentalization is required for successful symbiotic Ascomycota association with root cells, Mycological Research (2009), doi:10.1016/j.mycres.2009.02.013

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Fig 1 – Endophytic fungal hyphae showing formation of: (a) Type I – irregular cell shapes and (b) Type II – regular cell shapes in wheat root. Scale length depicted in red and width in green. (c) Showing different root-colonization pattern and deviation in fungus cell directions compared with root-cell membrane horizontal direction: (d) Type A – pronounced deviations in cells direction and e) Type B – without considerable deviation in cells direction (Dup and Ldown) compared with root axes (0) (arrows in green). Bar – 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Iregmin ¼ 1.2–1.6; thus, it was chosen for determining the range of Ireg variability in the two types (I and II). (2) Index of fungal cell directionality (Idir) describes fungus cell direction changes for individual hyphae inside a root (Fig 1c–e). Straight hyphae that grew aligned with the root axis were defined as the baseline pattern (0) in which the cell growth parallel to the root axis indicated not shifts in direction (Fig 1c). Fungus cells within an individual hypha whose growth axes deviated up (þ) or down () from baseline (0) were scored for each change in growth direction. Thus, Idir ¼ number of baseline cells/number of deviated cells  100, which resulted in a frequency scale of 5– 85 %. Based upon this scale, fungal cell direction was categorized in two types: Type A with Idir > 45 has cells aligned with the root axis, and Type B with Idir < 45 has deviated cell growth from the central root axis.

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Results The colonization pattern of two endophytic conidial ascomycetous strains, SMCD 2204 (class Dothidiomycetes) and SMCD 2213 (class Eurotiomycetes), differed in colonization morphologies within living and killed root cells (Figs 2–4). In living roots, the endophyte hyphae swelled between septa, adopting rounded, globose and subglobose forms (Fig 5a). In killed roots, hyphae were essentially cylindrical with at most variations in septum spacing (Fig 5b). In living root, SMCD 2204 colonization was inter- and intracellular (Fig 2a) with formation of intracellular hyphal coils (Fig 6a). The form of fungal cells within single root was of Type I with changing in cells regularity index. High irregularity in fungal cell form within single hypha (Fig 1a) was

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Fig 2 – Distribution of SMCD 2204 strain hyphae into the (a) living and (b) dead wheat root cells showing inter- and intra-cellular colonization pattern, whereas SMCD 2213 strain showing only intracellular colonization in both (c) living and (d) dead wheat root cells.

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observed going from Iregmin ¼ 1.22 to Iregmax ¼ 3.62 values, a range of Ireg ¼ 2.4 (Fig 4a). The Type A direction of cells (Fig 1d) was observed within simple hypha showing important growth deviation frequency Idir ¼ 80 % (þup and down) compared to central root axes (0) (Figs 4b, 5a). In killed root, SMCD 2204 was shown to have more abundant colonization in comparison to living root (Fig 7a,b) with both inter- and intra-cellular penetration patterns (Fig 2b).

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The fungal cells are of the Type II and there are no hyphal coils formed in dead cells (Fig 5b). The irregularity of cell form was much less than in living cells showing the Ireg indexes between 1.41 and 1.9 (Figs 1b, 4a) with a range of 0.49. The Type B cell growth had Idir ¼ 25 %, which is mostly growth parallel to the root axis (Figs 4b, 5b). The SMCD 2213 strain in association with living root was showing only intracellular (Fig 2c) colonization pattern. Also, hyphal knots (Fig 8a–c) and vesicles (Fig 6b) were produced within living root cells. The Type I-cell form was present and fluctuation of irregularity index varied between Iregmin ¼ 1.39 and Iregmax ¼ 3.45 (Figs 3a, 4a). The Ireg range was 2.06. Type A growth deviation was observed with relatively high Idir ¼ 65 % (Figs 4b, 5c). In the killed root, SMCD 2213 strain produced an abundant colonization (Fig 7c,d), whereas the penetration was intracellular (Fig 2d). Vesicles were also registered without visible knot structures. The index of cell irregularity was almost low (Fig 3b) and of the Type II, going from Iregmin ¼ 1.5 to Iregmax ¼ 2.1 and with a range of 0.6 (Fig 4a). The deviation of the Type B was observed and the fungal growth direction Idir ¼ 15 % (Figs 4b, 5d) followed the central root axes (0).

Discussion Fig 3 – Endophytic fungal hyphae in wheat root cells showing (a) irregularity – with changing fungal cell forms (b) and regularity – without changing forms. Note also fungal cell constricts at septal points associated with irregularity (arrow) (a) compared with regularity (arrow) (b).

The fungal endophytes include Ascomycota and Basidiomycota that colonize root tissues intracellularly and intercellularly (Jumpponen 2001). The mycobiont–root association varied from negative to neutral and positive measured by host performance or host tissue nutrient concentrations

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‘‘functional organ’’ to describe a specialized cell structure type comparable to an arbuscule being an organ for an arbuscular mycorrhiza. In this study, fungal cell form changes within single hyphae colonizing single root cells (Fig 1). In the Ascomycota, septa delimit compartments that can be further specialized for particular functions (Mims et al. 1988). In killed roots, endophyte hyphae had consistent hyphal diameter and cells had a consistent form, whereas there was considerable hyphal remodeling in the same endophyte species when growing in living roots. Kaminskyj & Hamer (1998) and Hodson et al. (in press) described an aberrant pattern in asexual Aspergillus nidulans hypha septation revealing that the fungal ‘‘swollen’’ cells must have undergone secondary enlargement following hyphal extension. According to Walther & Wendland (2004) the cellular form transition is also a key feature in the biology of some dimorphic fungal taxa where the actin cytoskeleton is required for this dimorphic switch. We used quantitative descriptors to compare and contrast the cell form based on hyphae septation pattern, cell form (Ireg) and cell growth direction (Idir) in living vs. killed roots. Fungus cell irregularity and direction showed considerably higher mean Ireg range and Idir values (Fig 4a,b) in the living cells compared with the dead cells. Iregmin values were similar in living and killed cells, however there were substantial differences in Iregmax and Idir values. Beside the hyphal colonization pattern changes, it seems important to better understand the cellular morphological changes within intracellular growing hyphae. Although conducted in vitro, our observation suggests that it is possible to assess the gradient of the continuum along the mutualism in leaving host cells to the saprotrophism in dead host cells – depending on plant physiological active vs. inactive environments with different availability of cell nutrition resources (Violi et al. 2007). Thus, the assessment of Ascomycota hyphal compartmentalization through changing in cell shape and direction (Fig 5) is seen as cornerstone for avoiding sometimes confusing ecological interpretation based on anatomical/morphological microscopic structure overlaps, usually reported in the context of superior Ascomycota and Basidiomycota (Lewis 1973; Jumpponen & Trappe 1998; Redman et al. 2001; Saikkonen et al. 1998) related with nutrient exchanges (Peterson et al. 2008) within active and inactive host cells. In this study, each fungal strain showed Type I-irregularity (symbiotic cell form) and Type A or three-dimensional direction (pattern of cell growth) within living cells (Figs 1a,d, 3a). In addition, Type II – regular cell forms and Type B – linear growth patterns were exclusively associated with inactive/ dead host cells (Figs 1b,e, 3b). We can suppose that the linear hyphal growth gives to fungi the ability to move relatively longer distances (Fig 7b,d) to explore for available food sources instead of waiting for the food coming through close mutualistic exchange found in interaction with the active plant cells. Under field conditions, saprotrophs must explore to acquire nutrients, whereas symbionts or parasite exploit a living interaction that presumably can supply ongoing nutrients. According to Bottone et al. (1998), hyphal growth in filamentous fungi proceeds in unidirectional radial pattern from a point of inoculation, and a secreted inhibitor protein absorbed by the advancing hyphae has account for that unidirectional growth. The results also showed that the higher diversity in formation

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Fig 4 – Fungal endophyte – (a) cell form alteration based on measured L/W and calculated Ireg-cell irregularity index. (b) Cell direction alteration based on measured frequency of direction changes: 0, D and L and calculated Idir-cell deviation index. Vertical bars represent standard errors of the means ( p < 0.05).

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(Declerck et al. 2005; Jumpponen 2001) attributable to variation between different fungal taxa and strains (Jumpponen & Trappe 1998). According to Redman et al. (2001) this association could represent a continuum ranging from parasitism to mutualism. However, incongruent data concerning colonization patterns have been reported due to the traditional or descriptive microscopic analyses based on simple comparison between endophytic structures or putative functional organs (coils, arbuscules or vesicles) produced by symbionts (Smith & Read 1997). In other words, it seemed that traditionally collected data were not fully informative on a quantitative basis, at least for Ascomycota, which show high morphological plasticity depending on environmental niche. According to Peterson et al. (2008), this group of endophytes may also morphologically mimic ectomycorrhiza or ectendomycorrhiza colonization patterns depending on plant or host species. In this study on Ascomycota, we showed that fungal colonization patterns are also host-life stage dependent. It seemed that further methodological advancement in microscopy is needed by combining ‘‘cell by cell’’ and ‘‘whole functional organ’’ data, to depict morphogenesis of multicellular hyphae. We are using

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Fig 5 – Endophytic hyphae in wheat root, visualized with lactofuchsin staining and confocal fluorescence microscopy: SMCD 2204 (a, b) and SMCD 2213 (c, d) illustrated changing hyphae direction in living wheat root cells (a, c) and without deviation or changing direction (b, d) in dead wheat root cells.

(Smith & Read 1997). Thus, their life is reduced to living plant cell niches for the accomplishment of a single obligatory biotrophic function. However, Glomeromycota have persisted for more than 400 million years and are found in >80 % of extant plant families. Thus, the specialization strategy adopted by the AM fungi, although obligatory, seems evolutionary very successful in terms of symbiotic relationship with plants.

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of functional organs in both tested strains was associated with the living cells compared with dead cells (Fig 7a,c). We believe that hyphal compartmentalization or cellular division in Ascomycota multiplied functions bringing much more plasticity to facultative biotrophs, thus, fulfilling more efficient multifunctional accomplishments as functional biotrophs. Glomeromycota mycorrhizal symbionts do not have multicellular hyphae

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Fig 6 – Endophytic SMCD 2204 hyphae visualized with lactofuchsin staining and confocal fluorescence microscopy showing formation of (a) coils and endophyte SMCD 2213 showed (b) vesicles (arrow). Please cite this article in press as: Abdellatif L et al., Endophytic hyphal compartmentalization is required for successful symbiotic Ascomycota association with root cells, Mycological Research (2009), doi:10.1016/j.mycres.2009.02.013

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685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 Fig 7 – SMCD 2204 (a, b) and SMCD 2213 (c, d) in wheat root showing different colonization patterns but the same pattern 716 Q11 of higher colonization frequency in dead roots (b, d) that of in living roots (a, c). Note also more regularity in fungal cell 717 direction associated with dead root cells (b, d) compared with pronounced regularity in living root cells. 718 719 Following penetration of living roots, endophytic hyphal root cortex are due to the phenomenon of equilibrated coloni720 growth was inter-, intra-cellular or both. These associations zation and/or lysis or digestion of hyphae by the host cells 721 might be limiting to one cell or situated in a limited area (Smith & Read 1997). Moreover, various patterns were ob722 around the penetration site (Sahay & Varma 1999). It might served in fungal cell abundance. The higher hyphal abun723 be that the low hyphal density settlements observed in living dance was more obvious in dead root (cortex and central 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 Fig 8 – SMCD 2204 strain penetration pattern in living wheat roots (a, c) showing (b) typical hyphal knots (arrows). 741 Note (a) early, (b) intermediate and (c) late stages in knot development.

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cells. Duckett & Ligrone (2005) suggested that the host cells do not accumulate lipid or other food reserves, as long as they contain healthy fungal coils. This present structure in the living root also explains the less frequency of colonization by fungus. Although some study has shown that the hyphal coils in a Paristype mycorrhiza interact with the host-cell cytoskeleton and are separated from the host-cell cytoplasm by an interfacial matrix forming an apoplastic compartment showing similarities to Arum-type mycorrhizae (Bonfante & Perotto 1995), it has yet to be shown that there is transfer of carbon compounds from host to fungus and phosphorus from fungus to host cells (Armstrong & Peterson 2002).

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856 857 858 859 860 861 862 863 864 865 866 867 868 Conclusions 869 870 Root endophytic fungi in wheat production may be used as 871 alternatives for chemical fertilizers and pesticides (Abdellatif 872 et al. 2007). They can induce beneficial morphological, physio873 logical and molecular changes in cereal hosts (Pesˇkan- Q8 874 Bergho¨fer et al. 2004), resulting in reprogrammed host-cell 875 tolerance to abiotic stresses, diseases resistance, and higher 876 yield (Waller et al. 2005). To elucidate the lifestyle of Ascomy877 cota endophytic fungi in wheat, we analyzed the symbiotic in878 teractions trough an endophytic development of hyphae 879 within root cortex of active vs. inactive host cells. In this study, 880 we assessed the colonization pattern of SMCD 2204 – class 881 Dothideomycetes and SMCD 2213 – class Eurotiomycetes pro882 posing a quantitative approach to assess endophyte morphol883 ogy in Ascomycota looking at the cellular level determinable 884 changes by using Ireg and Idir indexes. It is proposed to move 885 from traditional descriptive approach to a more objective mi886 croscopic approach in order to advance our comprehension 887 on the formation of functionally ‘‘complex structure’’ found 888 in ascomycetous endophytic and mycorrhizal root-symbionts 889 (Massicotte, 2005). This approach could be also expanded on 890 Basidiomycota as another multicellular fungal endophytic phylum, but unlike Glomeromycota mycorrhiza. 891 892 893 Uncited references 894 895 Bonfante and Giannazzi-Pearson, 1979; Fortin et al., 2002. 896 897 898 Acknowledgements 899 900 This research was financially supported by Natural Sciences 901 and Engineering Research Council of Canada Discovery Grant 902 and Agri-Food Innovation Found Chair (AFIF) in Microbial Bio903 technology & Bioproducts to VV, and Tunisia Ph.D. Research 904 Scholarship to LA. NSERC DG to SGWK. 905 906 references Q 9 907 908 909 Abdellatif L, Bouzid S, Vujanovic V, 2007. New plant growth910 promoting fungal endophytes reprogram wheat and provide 911 disease resistance. Plant Canada – Growth for the Future 912

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root cylinder) in comparison to living root (cortex tissues) (Fig 799 7b,e). In contrast, the colonization in living root cortex had 800 patchy distribution (Figs 7a,c, 8a) with an average of less 801 than 50 % of colonized living root cells (data not shown). 802 When compared with non-colonized living cells, colonized 803 cells showed hypertrophic reactions (Fig 8b) with increased 804 cell volume for 29.1 % when associated with SMCD 2204-inter805 cellular coil structures and 34.8 % in association with SMCD 806 2213-knot structures. Indeed, the relative increase in volume 807 was similar between two isolates. Changes in dead cell vol808 ume were not observed, consistent with saprotrophy. 809 An intracellular hyphal knot (HK) was described for the 810 first time as intracellular intertwining hyphal filaments, 811 which are surrounded by host-cell membrane (Fig 8). In eri812 coid mycorrhiza, some similar organs were described and 813 named as ‘‘complex structures’’ (Massicotte et al. 2005). Hod814 son et al. (in press) describe similar structures as sclerotium815 like hyphal aggregates. A sclerotium is a dormant asexual 816 resting organ that accumulates cytotoxic fungal metabolites 817 ensuring survival (Betina 1995) that typically is melanized. In 818 contrast, consistent with this report, Hodson et al. (in press) 819 showed that lactofuchsin-stained hyphal knots did fluoresce, which they interpreted as lack of substantial melanization. 820 We interpret hyphal knots as being formed by one or more 821 hyphae growing within a living root cell and actively interacting 822 with it. Hyphal HK’s likely differ from hyphal coils called ‘‘pelo823 Q6 tons’’ (Uetake et al. 1997), also intra-root cell structures often 824 found in Orchid and Ericoid mycorrhizal associations and 825 some biotrophic fungal infections (Massicotte et al. 2005). More826 over, knots diverged from pelotons – helical or spiral intracellu827 lar hyphae – by more ordered intertwining or braiding hyphae 828 forming knot-like hyphal aggregates (Fig 8a) with recognizable 829 surface motifs (Fig 8c). Like pelotons (Fig 6a), different stages 830 in colonization and collapse/digestion of the fungi are depicted 831 indicating mutualistic fungus–plant relationship. Beside these 832 interesting structures induced by host–fungus interactions in 833 active/inactive root cells, formation of vesicles has been also 834 registered (Fig 6b). With this result we can also better under835 stand the cellular events leading to the establishment of the mu836 tuality association pattern. In living root cell, the endophyte 837 performs a mutualistic relationship (Narisawa et al. 1998; Waller 838 et al. 2005) in which the fungi colonizes the plant root by ex839 changing nutrients with subsequent beneficial activities (Peter840 son et al. 2008; Selosse et al. 2004). It seemed that fungi in the 841 living tissues did not need to proliferate when they are satisfied 842 with their nutritional needs and exchanges with plant. Are resources allocated differently to symbiosis and reproduction? 843 Moreover, the apparent abundant hyphae and overall coloniza844 tion of dead root tissues as described allowing us a better under845 standing of the shift in fungal colonization pattern related with 846 inactive root cells, usually seen during root senescence stage 847 and formation of soil debris during the saprophytic stage of 848 these facultative biotrophs. In that stage, the fungus is using 849 the principal energy by the host as suggested by Peterson et al. 850 851 Q7 (1981). We also observed coils formed by SDMC 2204 strain and other cells packed hyphae with terminal vesicles formed by 852 SDMC2213 strain in wheat. Some other ascomycetous species 853 form both intracellular coils and ectomycorrhiza colonizing er854 icaceous roots (Piercey et al. 2002; Vra˚lstad et al. 2002). Interest855 ingly, the coiling structures were present only in living root

(Proceedings), Saskatoon, SK, Canada, pp. 137–139.

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