PERGAMON
Micron 33 (2002) 61±67
www.elsevier.com/locate/micron
Use of light and scanning electron microscopy to examine colonisation
of barley rhizosphere by the nematophagous fungus
Verticillium chlamydosporium
L.V. Lopez-Llorca*, J.J. Bordallo, J. Salinas, E. Monfort, M.L. LoÂpez-Serna
Departamento de Ciencias Ambientales y Recursos Naturales, Universidad de Alicante, Aptdo. Correos 99, 03080 Alicante, Spain
Received 24 May 2000; revised 6 September 2000; accepted 8 September 2000
Abstract
Barley roots were readily colonised by the nematophagous fungus Verticillium chlamydosporium. Light microscopy (LM) but also low
temperature scanning electron microscopy (LTSEM) revealed details of the colonisation process. Hyphae were found on the rhizoplane often
with dictyochlamydospores. Hyphae of V. chlamydosporium penetrated epidermal cells, often by means of appressoria. A hyphal network
was formed in epidermal and cortical cells. Likewise, hyphal coils were found within root cells next to transverse cell walls. Cortical cells
were the limits of fungal colonisation, since no hyphae were seen in the vascular cylinder. Modi®cations of root cell contents (phenolic
droplets and callose appositions) were common three weeks after inoculation with V. chlamydosporium. These features may indicate
induction of plant defence reactions in late stages of root colonisation by the fungus. Both LTSEM and LM have proved extremely useful
to describe root colonisation by the fungus. The results found may have implications in the mode action of nematophagous fungi against plant
parasitic nematodes. q 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Biocontrol; Rhizosphere colonisation; Nematophagous fungi; Cryosectioning; Low temperature scanning electron microscopy
1. Introduction
The rhizosphere is an important zone for the activity of
nematophagous fungi since plant-parasitic nematodes
commonly attack plant roots. Micro-organisms are found
in numbers 10±20 times (occasionally 100 times) higher
than in the root-free soil (Lynch, 1990). The presence of
nematode-trapping fungi in the rhizosphere of agricultural
plants have been studied by several authors (Peterson and
Katznelson, 1965; Gaspard and Mankau, 1986; Persmark
and Jansson, 1997). When comparing the natural occurrence
of nematode-trapping fungi in several rhizospheres, large
variations between plants was reported (Persmark and Jansson, 1997).
Persson and Jansson (1999), have recently found 9 out of
38 isolates of nematode trapping fungi, to be good colonisers of tomato rhizosphere. The capability to colonise root
was not advantageous for the biocontrol potential of these
isolates on root-knot nematode (Meloidogyne spp.), in
contrast to the egg-parasitic fungus V. chlamydosporium,
where root colonisation was suggested to be a prere* Corresponding author.
quisite for successful biological control (de Leij and
Kerry, 1991). Tomato with better root colonisation by
V. chlamydosporium than potato showed, when inoculated with the fungus and M. incognita, less egg infection (Bourne et al., 1996). This has been explained by
the large size of nematode galls in tomato roots, which
prevents eggs from exposure to soil and therefore infection. A strain of V. lecanii isolated from eggs of H.
glycines, has been found to colonise the soybean rhizosphere both in vitro and in pot tests, although was a
poor coloniser in the soil (Meyer et al., 1998).
In spite of the above, little is known on the root colonisation by nematophagous fungi at a cellular level. This rhizosphere phase is crucial for the nematophagous fungi
parasitising nematode eggs and females. The pathogens
are sedentary in roots where they induce profound cell
changes (Sijmons et al., 1994). Among them are the cyst
and root-knot nematodes which are amongst the most
important plant pathogenic species (Agrios, 1997).
In this paper, we show cellular details of root colonisation
by the egg parasite V. chlamydosporium using both light
microscopy as well as low temperature scanning electron
microscopy.
0968±4328/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0968-432 8(00)00070-6
62
L.V. Lopez-Llorca et al. / Micron 33 (2002) 61±67
2. Materials and methods
2.1. Plants and fungi
Barley (Hordeum vulgare L. var disticum) seed was
surface sterilised using 5% sodium hypochlorite (Prolabo,
France) with a drop of commercial detergent, for 30 min at
room temperature and shaking at 120 rpm. Seed were then
rinsed ®ve times (5 min each) in sterile distilled water.
Afterwards seed were dry blotted onto sterile ®lter paper.
Five to ten seeds were plated on germinating medium at
258C with a 12 h/12 h dark-light photoperiod. The medium
consisted of 1.2% agar added with glucose (10 g´l 21),
peptone (0.1 g´l 21) and yeast extract (0.1 g´l 21). Seedlings
free from contaminants were axenically placed in 30 ml
autoclaved tissue culture tubes (one per tube) (Sigma)
with 20 ml of water saturated vermiculite at 5±10 mm
from the surface. Tubes with seedlings were either left as
controls or were inoculated with V. chlamydosporium
(isolate V10, gift from Dr B. Kerry, IACR, Rothamsted,
UK). The fungus was inoculated in two ways: four 5 mm
diameter disks were either placed on the surface of the
vermiculite. Alternatively, disks were sunk 10 mm deep
and mixed with the vermiculite. To avoid root disturbances
fungus inoculation was carried out before seedling planting.
Tubes with seedlings or seedlings and fungus, were incubated as explained before for up to three weeks and were
sequentially sampled weekly.
2.2. Light microscopy (LM)
Two barley plants were selected at sampling time (1, 2
and 3 weeks after planting) and for each type of inoculation.
Fresh straight roots were chosen (two per plant). These were
cut in 0.5 cm pieces descending from the base of the stem to
the root apex.
Root pieces were embedded in OCT (Jung Tissue freezing medium, Leica, Germany) placed in a rubber ring (width
1 mm) of 1 cm height and of the same diameter. The
embedded roots were frozen at 2208C in a Leica CM1510
cryostat and the frozen blocks containing root fragments
were kept at 2208C before sectioning in the same device.
Roots were sectioned longitudinally as 50 mm wide
sections. Freshly cut sections were placed individually on
pre-cooled (2208C) microscopy slides using cool tweezers.
Before staining, sections were soaked in 2±3 drops of
distilled water and dislodged from the OCT embedding
medium. Sections were kept in water for 5 min at room
temperature. Water was then removed from the sections
which were stained by adding a drop of 0.01% w/v Toluidine Blue O (Panreac, Barcelona, Spain) in 0.1 M Potassium
dihydrogen phosphate±NaOH buffer pH 6. After placing a
coverslip, sections were kept at room temperature for 4±8 h
before observation to improve staining.
Sections were stored at 48C, but never longer than 12 h
Fig. 1. Light microscopy (LM) of longitudinal sections through uninoculated barley roots. (A) General view of 1 week old root. Bar size: 60 mm.
before observation. Samples were observed and photographed in an Olympus CH microscope.
2.3. Low temperature scanning electron microscopy
(LTSEM)
Specimens were processed as for light microscopy (see
embedding). Then they were ªcryotrimmedº in the cryostat
to the required level (Lopez-Llorca and Duncan, 1991).
Then they were frozen in sub-cooled liquid N2 in an Oxford
cryoSEM model CT 1500C attached to a Hitachi S-3000N
Scanning Electron Microscope. Samples were observed
uncoated and frozen (21508C), then etched (2908C) for
the time required to remove surface ice. Etched samples
were Au-coated in the Oxford cryoSEM model CT 1500C
sputter. Coated specimens were observed and images digitally recorded in the S-3000N SEM.
3. Results
When sectioned with the cryostat, barley roots (uninoculated) had the standard appearance of monocot roots in light
microscopy, the vascular cylinder was deeply stained with
toluidine blue, unlike the rest of the tissues (Fig. 1A). Under
higher magni®cation, epidermal cells were slender with
round endings in longitudinal section. Cortical cells adjacent to vascular cylinder were barrel-shaped.
Barley roots were readily colonised by Verticillium chlamydosporium. Two weeks after seedling inoculation, a
fungal mantle had developed on the rhizoplane, involving
epidermal cells. The fungal mantle was extensive on some
parts of the rhizoplane, as much as 20 mm in depth on occasions (not shown). As viewed by LTSEM, the rhizoplane
showed a mantle of mucilage with hyphae and root hairs
(Fig. 2A). Hyphae were often found running along the rhizoplane following grooves between adjacent epidermal cells.
Although rare, globose and deeply stained structures resembling conidia were sometimes found together with sharp
L.V. Lopez-Llorca et al. / Micron 33 (2002) 61±67
63
Fig. 2. LM and LTSEM of barley rhizoplane colonisation by V. chlamydosporium: (A) mucilage on rhizoplane with root hairs and hyphae (LTSEM); (B)
conidia and conidiophores on epidermal cells (LM); (C) close up of mature chlamydospores on root surface (LM). Bar sizes: 50 mm (A), 30 mm (B, C).
phialidic conidiophores on the rhizoplane (Fig. 2B). Resting
spores of the fungus (dictyochlamydospores) in different
developmental stages were also observed. Although very
common on the rhizoplane, sometimes they seemed to be
produced within epidermal cells. Chlamydospores were
very abundant three weeks after inoculation (Fig. 2C).
Hyphae of V. chlamydosporium were sometimes found
penetrating root hairs but more often epidermal cells
where appressoria were clearly seen (Fig. 3A, arrow).
Appressoria were also formed intracellularly (Fig. 3B).
When the fungus penetrated epidermal cells, an intracellular
network of hyphae was formed. This structure was both
revealed by LTSEM (Fig. 4A) as well as LM (Fig. 4B and
C). Commonly longitudinal cell wall were penetrated by
hyphae (Fig. 4A). The network was both intra as well as
intercellular. Intercellular hyphae often formed ªstepsº
following cell walls and thus rapidly reached inner parts
of the root (Fig. 4C). Evidence of fungal growth within
walls was found with LTSEM (Fig. 4D).
Hyphal coils were found within root cells. Coils were
seen in both epidermal (Fig. 5A) and cortical cells
(Fig. 5B), next to transverse cell walls, sometimes highly
Fig. 3. Penetration of barley root epidermal cells by V. chlamydosporium: (A) external penetration of epidermal cell with appressorium formation (arrow)
(LM); (B) penetration of adjacent epidermal cells by means of appressoria (arrow) (LM). Bar size: 30 mm.
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L.V. Lopez-Llorca et al. / Micron 33 (2002) 61±67
Fig. 4. Formation of hyphal network in epidermal cells of barley root by V. chlamydosporium: (A) general view of hyphae crossing cell walls (LTSEM); (B)
branch joining parallel hyphae to establish a network (LM); (C) typical ªstepsº in the development of the network (LM). Note apressoria when walls are
penetrated; (D) detail of a hypha penetrating (arrow) a cortex cell wall (LTSEM). Note undergrowth of the hyphae (arrow). Bar sizes: 50 mm (A), 30 mm
(B,C), 5 mm (D).
developed with several loops. LTSEM revealed within cells,
loops (Fig. 5C) which appeared to result from unsuccessful
wall penetrations.
Nuclei of epidermal cells, which were well stained by
toluidine blue, were sometimes colonised by the fungus
(Fig. 6A). LTSEM showed fungi in deep cortical cells but
not in phloem tissue (Fig. 6B). Similarly the fungus did not
reach xylem vessels.
Modi®cations of root cell contents by V. chlamydosporium were found, more so three weeks after inoculation.
They varied in appearance. Sometimes they looked like
small droplets free in the cytoplasm and staining with toluidine blue (Fig. 7A). In other instances dark cell wall appositions were viewed with LM (Fig. 7B). These structures were
often associated with hyphae penetrating cell walls. At this
time hyphae appeared lysed and their contents vacuolated
(Fig. 7C).
4. Discussion
In this paper we have clearly shown that V. chlamydosporium is able to colonise roots of barley plants. This
research is, to the best of our knowledge, the ®rst full report
describing in detail the microscopical details of root colo-
nisation by a nematophagous fungus. Similar work has
mainly been based on culture techniques to estimate rhizosphere colonisation by egg parasites (Bourne et al., 1996;
Meyer et al., 1998) and nematode-trapping fungi (Peterson
and Katznelson (1965), Gaspard and Mankau (1986), Persmark and Jansson (1997) and Persson and Jansson, 1999).
We have employed a versatile experimental protocol
involving cryo®xation, which can be used for generating
specimens ªcryotrimmedº that can be visualised with
LTSEM (Lopez-Llorca and Duncan, 1991). Alternatively,
sections in the cryostat can be obtained and processed for
light microscopy. Our staining protocol does neither
involves the use of ®xatives solutions (Gams et al., 1987)
nor the use of root tissue clearing techniques to visualise the
hyphae, an approach commonly used when working with
mycorrhizae (Brundett et al., 1994). Instead, we have optimised the root staining with toluidine blue in a buffer solution to differentially stain plant and fungus cell walls.
V. chlamydosporium grew on barley rhizoplane. Hyphae
sometimes followed epidermal cell grooves, a region where
rhizodeposition is particularly high. Conidia were rarely
seen but since they are produced on very delicate phialides
(Gams, 1988) they might have been lost when embedding
roots. However, chlamydospores were much more abundant
especially three weeks after inoculation. Since these resting
L.V. Lopez-Llorca et al. / Micron 33 (2002) 61±67
65
Fig. 5. Formation of hyphal coils in barley root cells by V. chlamydosporium: (A) multiple coils next to tranverse walls of epidermal cells (LM); (B) coils in
early cortex cells(LM); (C) early coils within epidermal cell (LTSEM). Bar sizes: 30 mm (A, B), 20 mm (C).
Fig. 6. Limits of barley root colonisation by V. chlamydosporium and other features: (A) nucleus colonisation by the fungus (LM); (B) cortex showing hyphae,
adjacent to vascular cylinder, where no fungus growth is found (LTSEM). Bar sizes: 30 mm (A), 50 mm (B).
spores are found in soils suppressive to cereal cyst nematode
Heterodera avenae (Crump and Kerry, 1981), the rhizosphere, together with infected nematodes, could be an
important source of inoculum ensuring the persistence of
the fungus in soil.
Using both LM and LTSEM, clear evidence of direct
penetration of root cells by the fungus was seen. V. chlamydosporium formed abundant appressoria just as has been
described for mycorrhizal fungi (San Antonio, 1990).
The fungus also crossed and even grew within epidermal
and cortical cell walls without any visible alterations of the
plant cell, indicating that V. chlamydosporium may at ®rst
not be recognised as a pathogen by barley root cells. Similar
results have been found for mycorrhizae (Gianinazzi-Pearson, 1996) and non-pathogenic endophytes (Sivasithamparam, 1998).
After root penetration, V. chlamydosporium mainly
spread longitudinally along root cells and hyphae
branched forming T shaped structures and forming a
network. Similar behaviour has been found in the colonisation of wheat roots by the pathogen Gaeumannomyces
graminis (syn. Ophiobolus graminis) (Garrett, 1970).
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L.V. Lopez-Llorca et al. / Micron 33 (2002) 61±67
Fig. 7. Modi®cations of barley root cell contents by V. chlamydosporium, 2 and 3 weeks after inoculation: (A) small droplets free in the cytoplasm stained by
toluidine blue (LM); (B) big size cell wall appositions (LM); (C) hyphae lysed with their contents vacuolated (LM). Bar size: 30 mm.
Intracellular hyphal coils were formed close to cell ends
by V. chlamydosporium in both epidermal and cortex
cells. Similar structures are formed by VAM fungi in
near cortex cells (Smith and Read, 1997) where they are
thought to have a nutritional role (Isaac, 1992). Nuclei
were sometimes colonised by V. chlamydosporium. This
fungal±plant cell interaction is unknown but deserves
further investigation.
V. chlamydosporium was con®ned to epidermis and cortical cells. It would therefore share a similar habitat as mycorrhizal (Smith and Read, 1997) or some cortical fungi that
antagonise fungal root pathogens (Sivasithamparam, 1998)
Three weeks after inoculation, two main alterations in the
normal appearance of root cells were observed. The droplets
because they stained with toluidine blue they are probably
phenolic in nature (Gollotte et al., 1993). The dark plant cell
wall appositions are probably callose deposits. Callose
appositions have been formed in wheat root cells as a
response to infection by G. graminis and are considered to
be a common plant response to pathogen invasion (Aist and
Israel, 1986; Isaac, 1992). Further research should be carried
out on the composition and dynamics of formation of these
structures by V. chlamydosporium. Occasional signs of
hyphal distortion (vacuolation) sometimes might represent
a late response of the roots to fungus colonisation, ®tting the
nature of fungus endophyte±host interaction as a balanced
antagonism (Schulz et al., 1999). The study of possible root
cell alterations due to V. chlamydosporium colonisation is
under progress in our laboratory.
Acknowledgements
Thanks are due to Mr A. AmoroÂs for help with Scanning
Electron Microscopy. We thank technical assistance by
M.L. Lorenzo. This research was funded by a Research
Grant FAIR5-PL97-3444 ªA strategy of Biomanagement
of root knot nematodesº of the European Union.
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