mycological research 113 (2009) 782791

journal homepage: www.elsevier.com/locate/mycres

Endophytic hyphal compartmentalization is required for

successful symbiotic Ascomycota association with root cells
Lobna ABDELLATIFa,c, Sadok BOUZIDc, Susan KAMINSKYJb, Vladimir VUJANOVICa,*
a

Department of Food and Bioproduct Sciences, University of Saskatchewan, 51 College Drive, Saskatoon, SK S7N 5A8, Canada
Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK S7N 5E2, Canada
c
Department of Plant Biotechnology, University of Tunis El Manar, 1060 Tunis, Tunisia
b

article info

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

Published online 6 March 2009

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

Corresponding Editor: John Dighton

morphologies during endophytic development of hyphae within the cortex of living vs.
dead root cells. Confocal laser scanning microscopy (CLSM) was used to characterize fungal

Keywords:

cell morphology within lactofuchsin-stained roots. Cell form regularity Ireg and cell growth

Ascomycota

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

Cell compartmentalization

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

Cell morphology

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

Fungal endophytes

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

Root

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

Symbiosis

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

Triticum turgidum

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.
2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction
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

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.: 1 306 966 5048.

E-mail address: vladimir.vujanovic@usask.ca
0953-7562/$ see front matter 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2009.02.013

Endophytic hyphal compartmentalization

reproduction cycle, were consequences of the life stages of

colonized plant organs, that is, they were affected by association with living vs. dead tissue. Biological significance of the
root cortex cell death in wheat on proliferation of ascomycetous weakly pathogenic Cochliobolus and avirulent Phialophora
isolates has also been suggested (Addy et al. 2005; Deacon &
Henry 1981; Deacon & Lewis 1982). In either case, establishment of the parasitic or mutualistic interaction is the result
of a highly sophisticated cross-talk between the partners
(Schafer et al. 2007). Hadacek & Kraus (2002) speculated that
fungal morphology changes may possibly be related to chemical variation specifically in the type of carbohydrates present
in the host cell. Whether root-cell structural changes (volume)
or carbohydrate changes in dead cells (associated with decomposition) are involved in fungal morphogenesis is still
unclear.
In this study, we hypothesized that the mutualistic pressure, two way fungus plant interactions, may differently affect the endophytic structures formed in living roots
compared to those in dead roots. If so, the same endophyte
might have different cell morphologies before and after root
senescence. Similarly, endophyte fungi might adopt different
colonization patterns depending on the metabolic activity of
the host plant cell tissue.
The aim of this study was therefore to compare Ascomycete endophyte colonization patterns and morphologies in
living and killed roots. To prevent major changes in killed
roots, i.e. cell membrane or volume modifications and arrangement deficiency, we used low-dose g-irradiation to ensure no shifting in root-inactive cell structural forms (Yu &
Wang 2006.). According to Natarajan & Kesavan (2005) and
Geraskin et al. (2007), g-irradiated barley meristematic cells
remain biochemically unchanged, so their influence on endophytic structural formation changes should be minimal.
Here, we describe fungusroot interactions in living and
killed roots.
SMCD* Saskatchewan Microbial Collection and Database,
College of Agriculture & Bioresources, University of Saskatchewan, SK, Canada.

Materials and methods

Two endophytic Ascomycota mitosporic strains (Kiffer &
Morelet 2000) SMCD 2204 (class Dothideomycetes) and SMCD
2213 (class Eurotiomycetes) (sensu Hibbet et al. 2007) isolated
from root of durum wheat Triticum turgidum L. (Saskatchewan,
Canada) were used in this study.

Endophyte growth experiments

Seeds were surface-sterilized with 95 % ethanol for 10 s,
rinsed in sterile distilled water (SDW) for 10 s, then submerged
for 3 min in 5 % sodium hypochlorite, rinsed 3 times in SDW,
and placed on potato dextrose agar (PDA) for germination.
Ten seeds were spread over 9 cm Petri plates. After 3 d of incubation (Precision Fisher Scientific Inc., Incubator MDL3EG) at
21  C in darkness, half of the young seedlings were co-cultured in association with fungal mycelia (5 mm2) for 4 d. Half
of the 3 d old seedlings were removed from the medium, their

783

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).

Microscopy
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
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 Students 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 (14), 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 Iregmin 1.21.6; thus, it was chosen

784

L. Abdellatif et al.

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.)

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 1ce). 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 () or down () from
baseline (0) were scored for each change in growth
direction. Thus, Idir number of baseline cells/number
of deviated cells  100, which resulted in a frequency scale
of 585 %. 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.

Results
The colonization pattern of two endophytic conidial ascomycetous strains, SMCD 2204 (class Dothideomycetes) and SMCD
2213 (class Eurotiomycetes), differed in colonization morphologies within living and killed root cells (Figs 24). 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

Endophytic hyphal compartmentalization

785

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.

observed going from Iregmin 1.22 to Iregmax 3.62 values,

a range of Ireg 2.4 (Fig 4a). The Type A direction of cells (Fig
1d) was observed within simple hypha showing important
growth deviation frequency Idir 80 % (up 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).

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 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 8ac) 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 1.39 and Iregmax 3.45 (Figs 3a, 4a). The Ireg range
was 2.06. Type A growth deviation was observed with relatively high Idir 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 1.5 to Iregmax 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 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 mycobiontroot association varied
from negative to neutral and positive measured by host performance or host tissue nutrient concentrations (Declerck et al.

786

L. Abdellatif et al.

Mean Ireg (L/w) Index Values

a 4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Living root

Dead root

Living root

SMCD 2204

Mean Idir-Index frequency (%)

Dead root

SMCD 2213

90
80

SMCD 2204

70
60

SMCD 2213

50
40
SMCD 2204

30
20

SMCD 2213

10
0
Living

Dead

ROOT CELLS
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).

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 functional organ to describe

a specialized cell structure type comparable to an arbuscule being an organ for an arbuscular mycorrhiza (Fortin et al. 2002). 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

Endophytic hyphal compartmentalization

787

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.

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
(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.
Following penetration of living roots, endophytic hyphal
growth was inter-, intra-cellular or both. These associations

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).

788

L. Abdellatif et al.

Fig 7 SMCD 2204 (a, b) and SMCD 2213 (c, d) in wheat root showing different colonization patterns but the same pattern
of higher colonization frequency in dead roots (b, d) than that of living roots (a, c). Note also there is more regularity in fungal
cell direction associated with dead root cells (b, d) compared with pronounced regularity in living root cells.

might be limiting to one cell or situated in a limited area

around the penetration site (Sahay & Varma 1999). It might
be that the low hyphal density settlements observed in living
root cortex are due to the phenomenon of equilibrated colonization and/or lysis or digestion of hyphae by the host cells

(Smith & Read 1997). Moreover, various patterns were observed in fungal cell abundance. The higher hyphal abundance was more obvious in dead root (cortex and central
root cylinder) in comparison to living root (cortex tissues)
(Fig 7b,e). In contrast, the colonization in living root cortex

Fig 8 SMCD 2204 strain penetration pattern in living wheat roots (a, c) showing (b) typical hyphal knots (arrows).
Note (a) early, (b) intermediate and (c) late stages in knot development.

Endophytic hyphal compartmentalization

had patchy distribution (Figs 7a,c, 8a) with an average of less

than 50 % of colonized living root cells (data not shown).
When compared with non-colonized living cells, colonized
cells showed hypertrophic reactions (Fig 8b) with increased
cell volume for 29.1 % when associated with SMCD 2204-intercellular coil structures and 34.8 % in association with SMCD
2213-knot structures. Indeed, the relative increase in volume
was similar between two isolates. Changes in dead cell volume were not observed, consistent with saprotrophy.
An intracellular hyphal knot (HK) was described for the
first time as intracellular intertwining hyphal filaments,
which are surrounded by host-cell membrane (Fig 8). In ericoid mycorrhiza, some similar organs were described and
named as complex structures (Massicotte et al. 2005). Hodson et al. (in press) describe similar structures as sclerotiumlike hyphal aggregates. A sclerotium is a dormant asexual
resting organ that accumulates cytotoxic fungal metabolites
ensuring survival (Betina 1995) that typically is melanized. In
contrast, consistent with this report, Hodson et al. (in press)
showed that lactofuchsin-stained hyphal knots did fluoresce,
which they interpreted as lack of substantial melanization.
We interpret hyphal knots as being formed by one or more
hyphae growing within a living root cell and actively interacting
with it. Hyphal HKs likely differ from coils called pelotons
(Uetake et al. 1997), also intra-root cell structures often found
in Orchid and Ericoid mycorrhizal associations and some biotrophic fungal infections (Massicotte et al. 2005). Moreover, knots diverged from pelotons helical or spiral intracellular hyphae by
more ordered intertwining or braiding hyphae forming knot-like
hyphal aggregates (Fig 8a) with recognizable surface motifs
(Fig 8c). Like pelotons (Fig 6a), different stages in colonization
and collapse/digestion of the fungi are depicted indicating mutualistic fungusplant relationship. Beside these interesting
structures induced by hostfungus interactions in active/inactive root cells, formation of vesicles has been also registered
(Fig 6b). With this result we can also better understand the cellular events leading to the establishment of the mutuality association pattern. In living root cell, the endophyte performs
a mutualistic relationship (Narisawa et al. 1998; Waller et al.
2005) in which the fungi colonizes the plant root by exchanging
nutrients with subsequent beneficial activities (Peterson et al.
2008; Selosse et al. 2004). It seemed that fungi in the living tissues
did not need to proliferate when they are satisfied with their nutritional needs and exchanges with plant. Are resources allocated differently to symbiosis and reproduction? Moreover, the
apparent abundant hyphae and overall colonization of dead
root tissues as described allowing us a better understanding of
the shift in fungal colonization pattern related with inactive
root cells, usually seen during root senescence stage and formation of soil debris during the saprophytic stage of these facultative biotrophs. In that stage, the fungus is using the principal
energy by the host as suggested by Peterson et al. (1981). We
also observed coils formed by SMCD2204 strain and other cells
packed hyphae with terminal vesicles formed by SMCD2213
strain in wheat. Some other ascomycetous species form both intracellular coils and ectomycorrhiza colonizing ericaceous roots
(Piercey et al. 2002; Vralstad et al. 2002). Interestingly, the coiling
structures were present only in living root cells. Duckett &
Ligrone (2005) suggested that the host cells do not accumulate
lipid or other food reserves, as long as they contain healthy

789

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 Paris-type 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).

Conclusions
Root endophytic fungi in wheat production may be used as
alternatives for chemical fertilizers and pesticides (Abdellatif
et al. 2007). They can induce beneficial morphological, physiological and molecular changes in cereal hosts (PeskanBerghofer et al. 2004), resulting in reprogrammed host-cell
tolerance to abiotic stresses, diseases resistance, and higher
yield (Waller et al. 2005). To elucidate the lifestyle of Ascomycota endophytic fungi in wheat, we analyzed the symbiotic interactions trough an endophytic development of hyphae
within root cortex of active vs. inactive host cells. In this study,
we assessed the colonization pattern of SMCD 2204 class
Dothideomycetes and SMCD 2213 class Eurotiomycetes proposing a quantitative approach to assess endophyte morphology in Ascomycota looking at the cellular level determinable
changes by using Ireg and Idir indexes. It is proposed to move
from traditional descriptive approach to a more objective microscopic approach in order to advance our comprehension
on the formation of functionally complex structure found
in ascomycetous endophytic and mycorrhizal root-symbionts
(Massicotte et al. 2005). This approach could be also expanded
on Basidiomycota as another multicellular fungal endophytic
phylum, but unlike Glomeromycota mycorrhiza.

Acknowledgements
This research was financially supported by Natural Sciences
and Engineering Research Council of Canada Discovery Grant
and Agri-Food Innovation Found Chair (AFIF) in Microbial Biotechnology & Bioproducts to VV, and Tunisia Ph.D. Research
Scholarship to LA. NSERC DG to SGWK.

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