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Annals of Applied Biology ISSN 0003-4746
RESEARCH ARTICLE
Colonisation of barley roots by endophytic Fusarium equiseti
and Pochonia chlamydosporia: Effects on plant growth
and disease
J.G. Maciá-Vicente1 , L.C. Rosso2 , A. Ciancio2 , H.-B. Jansson1 & L.V. Lopez-Llorca1
1 Laboratory of Plant Pathology, Department of Marine Sciences and Applied Biology, Multidisciplinary Institute for Environmental Studies (MIES) Ramón
Margalef, University of Alicante, Apto 99, 03080 Alicante, Spain
2 Istituto per la Protezione delle Piante, CNR, Via Amendola 165/A, 70126, Bari, Italy
Keywords
Biological control; Fusarium equiseti;
Gaeumannomyces graminis var. tritici; plant
growth promotion; Pochonia chlamydosporia;
root endophytes; take-all.
Correspondence
J.G. Maciá-Vicente, Laboratory of Plant
Pathology, Department of Marine Sciences
and Applied Biology, Multidisciplinary Institute
for Environmental Studies (MIES) Ramón
Margalef, University of Alicante, Apto 99,
03080 Alicante, Spain.
Email: [email protected]
Received: 12 September 2008; revised version
accepted: 26 June 2009.
doi:10.1111/j.1744-7348.2009.00352.x
Abstract
Colonisation of plant roots by endophytic fungi may confer benefits to the host
such as protection against abiotic or biotic stresses or plant growth promotion.
The exploitation of these properties is of great relevance at an applied level,
either to increase yields of agricultural crops or in reforestation activities.
Fusarium equiseti is a naturally occurring endophyte in vegetation under
stress in Mediterranean ecosystems. Pochonia chlamydosporia is a nematode eggparasitic fungus with a worldwide distribution. Both fungi have the capacity to
colonise roots of non-host plants endophytically and to protect them against
phytopathogenic fungi under laboratory conditions. The aim of this study
was to evaluate the root population dynamics of these fungi under nonaxenic practical conditions. Both fungal species were inoculated into barley
roots. Their presence in roots and effects on plant growth and incidence
of disease caused by the pathogen Gaeumannomyces graminis var. tritici were
monitored periodically. Both fungi colonised barley roots endophytically over
the duration of the experiment and competed with other existing fungal root
colonisers. Furthermore, colonisation of roots by P. chlamydosporia promoted
plant growth. Although a clear suppressive effect on disease could not be
detected, F. equiseti isolates reduced the mean root lesion length caused
by the pathogen. Results of this work suggest that both F. equiseti and
P. chlamydosporia are long-term root endophytes that confer beneficial effects to
the host plant.
Introduction
Endophytism is a ubiquitous phenomenon that occurs
in all plants and environments. Its importance in plant
development and distribution is starting to be unravelled. Fungal endophytes may help their host plants
adapt to habitats, protect against biotic or abiotic stresses,
promote plant growth or facilitate soil nutrient uptake
(Sieber, 2002; Rodriguez et al., 2004; Schulz & Boyle,
2005; Rodriguez & Redman, 2008). In studies of root
endophytism in natural plant communities under stress
(Maciá-Vicente et al., 2008a), a large component of fungal
root endophytes antagonistic to important root pathogens
Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors
Journal compilation © 2009 Association of Applied Biologists
such as wilt and take-all fungi was found (Maciá-Vicente
et al., 2008b). Bona fide biocontrol fungi, which include
important antagonists of plant pests and diseases (e.g.
nematophagous and entomopathogenic fungi), can also
behave endophytically (Lopez-Llorca et al., 2006).
Fusarium equiseti, a naturally occurring endophyte from
vegetation under stress in Mediterranean ecosystems,
can also colonise non-host roots (Maciá-Vicente et al.,
2008a,b). This species produces toxins antagonistic to
fungal pathogens and plant parasitic nematodes (Nitao
et al., 2001; Horinouchi et al., 2007; Maciá-Vicente et al.,
2008b). Fusarium equiseti also controls parasitic plants
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Root colonisation by endophytes and effects on host plant
(Kirk, 1993). Pochonia chlamydosporia is a nematode eggparasitic fungus and occurs widely in cyst and root-knot
nematode-infested soils around the world (Kerry, 1993).
Pochonia chlamydosporia also parasitizes economically
important phytopathogenic fungi, including the take-all
fungus Gaeumannomyces graminis var. tritici (Leinhos &
Buchenauer, 1992; Ehteshamul et al., 1994; Jacobs et al.,
2003; Monfort et al., 2005). All these properties make
F. equiseti and P. chlamydosporia as potential biocontrol
agents for both phytopathogenic fungi and plant parasitic
nematodes.
The plant benefits of endophytism can be exploited
in agriculture (e.g. crop protection/adaptation or yield
increase) or in ecosystem restoration (e.g. plant adaptation to degraded environments). Practical use of the
endophytic capacities of selected fungi may come from the
use–in alternative hosts–of natural endophytes, which
confer benefits to their original hosts (Rodriguez et al.,
2008). Alternatively, beneficial fungi (e.g. biocontrol
fungi) with endophytic capabilities can be inoculated
in roots (Lopez-Llorca et al., 2006) or other plant tissues
(Gómez-Vidal et al., 2006; Vega, 2008). The successful
application of endophytes depends on our understanding
of their behaviour in the plant host and their interactions
with other rhizospheric microorganisms. This understanding includes the study of the dynamics of fungal
colonisation of plant tissues.
Colonisation of barley roots by F. equiseti and
P. chlamydosporia has already been characterised under
axenic laboratory conditions (Bordallo et al., 2002; LopezLlorca et al., 2002; Maciá-Vicente et al., 2008b, 2009).
However, there are to date no reports on their long-term
capacity to persist endophytically within roots of plants
growing in non-axenic conditions. Pochonia chlamydosporia has previously been inoculated into rhizospheric soil of
different crop plants, for control of both fungal pathogens
and nematode parasites (Bourne et al., 1996, 1998; Monfort et al., 2005). The presence of the fungus in plant
roots under non-axenic conditions was not reported in
these studies. This may be an important feature because
P. chlamydosporia seems to be a poor competitor when
introduced into the soil (Monfort et al., 2006).
We propose that root endophytism provides biocontrol agents targeted to soil pathogens (e.g. F. equiseti or
P. chlamydosporia) with an adaptative advantage by lowering competition with soil microbiota. Fungi within the
root can easily find nutrients and environmental conditions suitable for multiplication. In this way, endophytism
could create a stable source of inoculum to sustain the
populations of the microorganism in the rhizospheric
soil. The aim of this study was to understand the effects
of endophytic colonisation of barley roots by F. equiseti
and P. chlamydosporia on plant growth and disease. For
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J.G. Maciá-Vicente et al.
this purpose, we inoculated both fungal species in barley roots to ensure their endophytic development and
grew the inoculated plants under greenhouse conditions.
We monitored the presence of the two endophytes in
roots periodically, either in the rhizoplane or within the
roots. We also tested the effects of endophytic colonisation on plant growth. In a second experiment, we
included a fungal root pathogen and evaluated the influence of the endophytic development of F. equiseti and
P. chlamydosporia on the presence of the pathogen in the
roots and root disease. For this experiment, we selected
G. graminis var. tritici, the causal agent of take-all, a root
disease of cereal crops worldwide. Gaeumannomyces graminis var. tritici has been used extensively in root biology
studies and is a model pathogen for experimental systems
such as the one presented herein.
Materials and methods
Fungal material
Fusarium equiseti Corda (Saccardo) isolates, 10/3.3.1
(Fe10331) and 45/1.2.1 (Fe45121), and P. chlamydosporia
(Goddard) Zare & Gams isolates, 123 (Pc123) and INEMVC-21 (Pc21), were selected for inoculation experiments.
Fusarium equiseti Fe10331 was isolated from roots of
Corynephorus canescens (Poaceae) growing in a sandy soil
and Fe45121 from roots of Lygeum spartum (Poaceae)
growing in a salt marsh (Maciá-Vicente et al., 2008a).
Pochonia chlamydosporia Pc123 was isolated from Heterodera
avenae infected eggs in SW Spain (Olivares-Bernabeu &
Lopez-Llorca, 2002) and Pc21 from soil and egg masses
of Meloidogyne sp. infecting kiwifruit trees at Metaponto
(Italy). Gaeumannomyces graminis (Saccardo) Arx & Olivier
var. tritici Walker (Ggt) was kindly provided by Dr K.
Sivasithamparam, University of Western Australia, and
has been used previously for co-inoculation of barley
plants with Fe10331, Fe45121 and Pc123 under axenic
conditions (Monfort et al., 2005; Maciá-Vicente et al.,
2008b).
Inoculation of barley roots with fungal endophytes
Barley (Hordeum vulgare L. var. distichum) roots were
inoculated with four plugs (5 mm in diameter) of
Fe10331, Fe45121, Pc123 or Pc21 growing on corn meal
agar (CMA, BBL, Sparks, MD, USA) in tubes containing
sterilised vermiculite, as described in Maciá-Vicente et al.
(2008b). Control treatments consisted of tubes with plugs
of non-colonised CMA. Plants were placed in a growth
chamber with a photoperiod of 16/8 h (light/dark) at
23◦ C for 7 days. Plants were then transferred to 1 L pots
with pasteurised sandy soil from Ragusa (Italy). Pots were
placed in a greenhouse and irrigated periodically.
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J.G. Maciá-Vicente et al.
Five plants from each treatment were sampled at intervals of 7 (from tubes with sterilised vermiculite), 21,
35, 49 and 63 (from pots with soil) days after inoculation (dai). Fresh shoot and root weight per plant were
recorded upon sampling. Roots of each plant were then
split into two halves. One was left untreated whereas the
other one was surface sterilised in 1% sodium hypochlorite for 1 min, washed three times in sterile distilled
water (1 min each) and blotted onto sterilised filter
paper. Non-sterilised and surface-sterilised roots were cut
into approximately 1 cm fragments and six root pieces
per plant were plated onto CMA supplemented with
50 mg mL−1 streptomycin, 50 mg mL−1 penicillin G and
1% Triton X-100 (Sigma, St. Louis, MO, USA). Sterilised
root pieces were imprinted onto plates with the same
medium before plating to evaluate the efficacy of the
surface sterilisation method (Hallmann et al., 2006). After
5–7 days, fungal colonisation of root pieces was recorded,
and developing fungal colonies were isolated on potato
dextrose agar (PDA, Oxoid Ltd, Hampshire, UK) for identification. Percentage of root colonisation by endophytes
(F. equiseti and P. chlamydosporia) and other filamentous
fungi was then calculated as: Nd /Nt ·100, where Nd is the
number of root pieces from which the fungi were detected
and Nt the total number of root pieces.
Co-inoculation of barley roots with fungal root
endophytes and Ggt
Co-inoculation of barley roots with either Fe10331,
Fe45121, Pc123 or Pc21 and Ggt was performed as
described in Maciá-Vicente et al. (2008b). Control treatments were inoculated with Ggt only and included four
uncolonised plugs of CMA instead of the endophyte.
Inoculated plantlets were kept in a growth chamber with
the same conditions previously mentioned for 7 days, and
then transferred to 1 L pots with a non-sterilised sandy
soil from Arenal de Biar (SE, Spain; Monfort et al., 2006).
Ten plants per treatment were sampled at intervals of
7 (from tubes with sterilised vermiculite), 35 and 63 dai
(from pots with soil). Plants were rated for disease using
an arbitrary scale of 0–4 with intervals of 0.5 (Cotterill &
Sivasithamparam, 1987). Shoot and root fresh weight per
plant were recorded, and the root system of each plant
was then carefully scanned with an EPSON Expression
1680 Pro scanner (Seiko Epson, Nagano, Japan) for
further analysis. Root images were digitised with the
software Photoshop CS2 (Adobe); root length above
symptoms were coloured blue, symptoms red and root
length below symptoms green. Red–green–blue colours
of digitised images were detected separately using the
software analySIS (Soft Imaging System GmbH, Münster,
Germany). Total root length, mean lesion length and
Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors
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Root colonisation by endophytes and effects on host plant
percentage of effective root length (ERL) per plant (Aberra
et al., 1998) were then estimated.
Plant roots were either non-sterilised or surface
sterilised as described above and cut into approximately
1 cm long fragments. Ten root fragments per plant
were plated onto CMA supplemented with 50 mg mL−1
streptomycin, 50 mg mL−1 penicillin G and 1% Triton
X-100. After 5–7 days, colonisation of the root pieces was
recorded, and developing fungal colonies were isolated
on fresh PDA for fungal identification. Percentage of root
colonisation by endophytes, Ggt and other filamentous
fungi was calculated. Remaining root material was stored
at –76◦ C for further studies.
Polymerase chain reaction detection of Ggt
in barley roots
Because adequate quantification of Ggt in roots could not
be achieved by culturing techniques, its presence in roots
was assessed by polymerase chain reaction (PCR)-based
methods. Surface-sterilised root systems from plants sampled 63 dai were used for genomic DNA extraction.
Roots of three plants per treatment were selected randomly for this experiment. DNA was extracted from
surface-sterilised roots as follows: approximately 0.6–1 g
of root tissue was ground in liquid nitrogen and DNA
was extracted at 65◦ C for 1 h with 4 mL of DNA extraction buffer containing 100 mM Tris–HCl pH 8.4, 1.4 M
NaCl, 25 mM EDTA (Sigma), 2% CTAB (Sigma) and 2%
low weight polyvinylpyrrolidone (PVP, Sigma). Extracts
were purified with one volume phenol: chloroform:
isoamyl-alcohol (IAA)(25:24:1) and then with one volume chloroform: IAA (24:1) and precipitated in one
volume isopropanol. DNA pellets were washed twice in
70% ethanol, air-hood dried and resuspended in 1 × TNE
buffer. RNAse A (Sigma) was added to each treatment
and tubes were incubated for 30 min at 37◦ C. Extracts
were again purified in phenol: chloroform: IAA and chloroform: IAA and precipitated in two volumes absolute
ethanol. Pellets were again washed twice in 70% ethanol
and allowed to dry, and DNA was finally resuspended
in 1 × TE buffer. The extraction method used resulted in
DNA suspensions containing brown material, which could
be phenolic compounds such as lignin (Chen et al., 1996).
These compounds inhibited PCR amplification (data not
shown). Therefore, DNA extracts were further purified
using the GENECLEAN SPIN Kit (Qbiogene Inc., Carlsbad,
CA, USA) following the manufacturer’s instructions. Purified extracts were quantified using H6024 Hoechst stain
(Sigma) as described in Ausubel et al. (2002) and stored at
4◦ C until use. To perform positive controls for PCR reactions, genomic DNA from Ggt pure cultures was obtained
as in O’Donnell et al. (1998), with minor modifications.
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Primers NS5 (5 -AACTTAAAGGAATTGACGGAAG-3 )
and GGT-RP (5 -TGCAATGGCTTCGTGAA-3 ) were used
for PCR detection of Ggt in roots as in Fouly &
Wilkinson (2000), with modifications. Amplifications
were performed in a total volume of 50 μL, containing:
1 × Flexi buffer, 2 mM MgCl2 , 0.2 mM dNTP, 0.5 μM
each of the primers, 10 ng of DNA template and 2.5 units
of Taq DNA polymerase (Promega Corporation, Madison,
WI, USA). A positive control included 1 ng of Ggt genomic
DNA. A negative control, in which genomic DNA was
replaced by water, was used as a test for contamination.
Temperature cycling was carried out in a PTC-100
Thermal cycler (MJ Research, Waltham, MA, USA). An
initial denaturation step at 93◦ C for 3 min was followed
by 35 cycles of denaturation at 93◦ C for 1 min, annealing
at 57◦ C for 1 min and extension at 72◦ C for 1 min. A final
step at 72◦ C for 5 min was performed after the cycles.
An aliquot of 10 μL amplification products were loaded
onto a 2% electrophoresis agarose gel, stained with SYBR
Green I (Sigma) and photographed under ultraviolet
light. Bands in the gel corresponding to 410-bp Ggtspecific amplicon were excised, purified with Ultrafree
columns (Millipore Corp., Cork, Ireland) and sequenced
(Macrogen Inc., Seoul, Korea). Sequences amplified from
root extracts were compared with the sequence from Ggt
pure cultures and with those in the GenBank database
using BLAST analysis (Altschul et al., 1990).
Data analysis
Data were checked for normality using the Shapiro–Wilk
test, and Levene’s test was used to study homogeneity
of variance across groups. Data following a normal distribution were compared using two-way ANOVA for differences between treatments and/or sampling times. Nonnormal data were compared using the Kruskal–Wallis
(K–W) rank sum test. Either Student’s t-test or Wilcoxon
test with corrections for multiple testing was used for
pair-wise comparisons. In all cases, significance level considered was 95%. All analyses were performed using the
R 2.5.1 software (R Development Core Team, 2007).
Results
Rhizosphere dynamics of F. equiseti and P. chlamydosporia
Both F. equiseti and P. chlamydosporia occurred with a
high isolation frequency (from approximately 78% to
100% colonisation) in non-sterilised roots 7 dai, from
plants growing in tubes with vermiculite. Their occurrence decreased in roots after transplanting to pots with
soil (Fig. 1). Both fungal species were isolated at low frequency from surface-sterilised roots, but their presence
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J.G. Maciá-Vicente et al.
remained relatively constant with time (Fig. 1). Generally, the two isolates of each fungal species behaved
in a similar manner: Fe10331 and Fe45121 populations
decreased gradually in the ectorhizosphere, whereas their
endophytic populations were stable at around 7% and
20% colonisation. Colonisation at 63 dai reached higher
values than in non-sterilised roots at the same time
(Fig 1A and Fig. 1B). Pc123 and Pc21 had completely
colonised the outer root system 7 dai, as assessed by
plating of non-sterilised roots. However, its incidence
was reduced severely (from 100% to 0% for Pc123
and 7% for Pc21) after plants were transferred to pots
with soil (Fig. 1C and Fig. 1D). Isolation percentages of
both P. chlamydosporia isolates from surface-sterilised roots
were comparable with those of F. equiseti isolates 7 dai
(13%–23%), but decreased in subsequent samplings of
plants from pots (Fig. 1C and Fig. 1D).
The presence of both F. equiseti and P. chlamydosporia
as endophytes affected colonisation of barley roots by
other filamentous fungi. In treatments with endophytes,
the isolation of other fungi from surface-sterilised roots
decreased significantly (K–W, P < 0.05) when compared
with that of uninoculated controls, except at 49 dai
(Fig. 1). On the contrary, the percent colonisation
of filamentous fungi other than endophytes obtained
from non-sterilised roots was different between roots
inoculated with F. equiseti or P. chlamydosporia. Although
colonisation of the ectorhizosphere of roots inoculated
with either Fe10331 or Fe45121 with other fungi
increased gradually with time following a reduction
of F. equiseti populations, overall colonisation by other
fungi was significantly lower than that in controls
(K–W, P < 0.05; Fig. 1A, Fig. 1B and Fig. 1E). For roots
inoculated with P. chlamydosporia, isolation of other fungi
did not differ (K–W, P = 1) from control treatments: after
the transfer of the plants to pots with soil, colonisation
of the root by soil contaminants reached values of
83%–100% (Fig. 1C, Fig. 1D and Fig. 1E).
Effects of root colonisation by F. equiseti
and P. chlamydosporia on plant growth
Colonisation of roots by F. equiseti isolates had virtually no effect on plant growth (Fig. 2). On the contrary,
P. chlamydosporia had a clear plant growth-promoting
effect on barley (Fig. 2). Plants inoculated with either isolate Pc123 or Pc21 demonstrated a significant (ANOVA,
P < 0.05) increase in shoot weight with respect to controls from 49 dai onwards (Fig. 2A). At 63 dai, the shoot
weight of plants inoculated with either Pc123 or Pc21
was 1.6- and 1.9-fold that of uninoculated control plants,
respectively. This growth promotion by P. chlamydosporia
was also observed in roots, with a significant (ANOVA,
Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors
Journal compilation © 2009 Association of Applied Biologists
J.G. Maciá-Vicente et al.
Root colonisation by endophytes and effects on host plant
Figure 1 Dynamics of colonisation of barley roots inoculated with the endophytes (open circles) Fe10331 (A), Fe45121 (B), Pc123 (C) and Pc21 (D), and
by other indigenous soil filamentous fungi (solid circles), assessed in both non-sterilised (solid lines) and surface-sterilised (dashed lines) roots. Values of
colonisation by filamentous fungi other than Fusarium equiseti and Pochonia chlamydosporia in control (uninoculated) treatments are provided in plot E.
Bars show standard errors.
Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors
Journal compilation © 2009 Association of Applied Biologists
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J.G. Maciá-Vicente et al.
Figure 2 Effect of root colonisation by Fusarium equiseti and Pochonia chlamydosporia on barley growth: shoot fresh weight (A) and root fresh weight (B).
Bars show standard errors.
P < 0.05) root weight increase at 63 dai when compared
with controls (Fig. 2B). At this time, the rate of weight
increase with respect to the control plants was 1.9 for
Pc123 and 1.8 for Pc21. Furthermore, plants inoculated
with P. chlamydosporia presented an early development of
spikes at 63 dai, when compared with plants from the
other treatments (data not shown).
Rhizosphere dynamics of F. equiseti and P. chlamydosporia
in the presence of Ggt
The patterns of endophyte colonisation of Ggt-infected
barley roots differed from those of plants inoculated with
the endophytes only (Fig. 3). In this experiment, root
colonisation was scored at 7, 35 and 63 dai. Fe10331
displayed higher colonisation percentages, for both nonsterilised and surface-sterilised roots than in the previous
experiment, and significantly (K–W, P < 0.05) higher
colonisation rates than Fe45121 at 7 and 35 dai (Fig. 3A).
Fe45121 was isolated at lower levels from both nonsterilised and sterilised roots after 7 days of growth in
sterilised vermiculite, and from non-sterilised roots from
pots with soil at 35 dai (Fig. 3B), than in the experiment with the endophyte alone (Fig. 1B). Colonisation
by Fe10331 and Fe45121 of non-sterilised and surfacesterilised roots was not significantly different (K–W,
P > 0.05) at 63 dai (20%–44%). Pc123 followed the
same pattern of colonisation of the root as in the previous
experiment, but with higher colonisation rates (Fig. 3C).
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Pc21 showed a significantly higher (K–W, P < 0.05) isolation percentage than Pc123 (65% and 9%, respectively) in
the ectorhizosphere 35 dai. Isolation rates of both Pc123
and Pc21 at 63 dai did not differ significantly (K–W,
P > 0.05) in non-sterilised and in surface-sterilised roots
(9%–30%; Fig 3C and Fig 3D).
Quantification determined by culturing Ggt from
infected roots could only be achieved from plantlets
growing in culture tubes with sterilised vermiculite 7
dai. In this case, isolation rates ranged between 2 ± 0.6%
and 6 ± 1.3% in non-sterilised roots, with no significant
differences among treatments. In surface-sterilised roots,
values ranged between 0 ± 0% for Fe10331 and Pc21 and
4 ± 1.3% in control treatments, with no significant differences. At 35 and 63 dai, sterile dark-pigmented mycelia
were frequently recovered from roots of plants in all treatments, but these could not be related to Ggt either in CMA
or PDA culture media, because of a lack of sporulation
of the colonies. Therefore, the presence of Ggt in barley
roots was assessed by PCR using primers specific to Ggt. All
PCR reactions including DNA from barley roots inoculated
with Ggt only and those with both Ggt plus endophytes,
and with DNA extracted from Ggt pure cultures, yielded
amplification products. All amplification products showed
several unspecificities, but approximately 410-bp Ggtspecific amplicon was found in all treatments (data not
shown). PCR products from seven independent amplification reactions were purified from the gels and sequenced.
In all cases, sequences matched those obtained from Ggt
pure cultures and from GenBank databases.
Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors
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J.G. Maciá-Vicente et al.
Root colonisation by endophytes and effects on host plant
Figure 3 Dynamics of colonisation of barley roots inoculated with both Ggt and Fe10331 (A), Fe45121 (B), Pc123 (C) and Pc21 (D) endophytes (open
circles), and by other indigenous soil filamentous fungi (solid circles), assessed in both non-sterilised (solid lines) and surface-sterilised (dashed lines)
roots. Values of colonisation by filamentous fungi other than Fusarium equiseti and Pochonia chlamydosporia in control (Ggt only) treatments are provided
in plot E. Bars show standard errors.
Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors
Journal compilation © 2009 Association of Applied Biologists
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Isolation of fungal contaminants from surface-sterilised
roots was significantly lower (K–W, P < 0.05) only in
plants treated with Fe10331 than in controls with Ggt
alone 63 dai (from 31% to 8%, respectively), whereas
both Fe10331 and Pc21 reduced the occurrence of other
filamentous fungi in non-sterilised roots at 35 dai (from
89% to 52% and 54% respectively) and Fe10331 at 63
dai (from 31% to 8%; Fig. 3). Therefore, inhibition of
the presence of fungi other than the inoculated ones
seemed to be correlated with the population size of the
endophytes at each sampling time (Fig. 3).
Effects of root colonisation by F. equiseti and
P. chlamydosporia on take-all disease
No effect was found on fresh shoot and root weight either
in plants inoculated with F. equiseti or P. chlamydosporia
and Ggt with respect to control treatments with Ggt
only. Both P. chlamydosporia isolates Pc123 and Pc21
significantly increased (ANOVA, P < 0.05) the total
root length 7 dai (0.584 ± 0.033 and 0.577 ± 0.039 m,
respectively) when compared with the other treatments
(0.430 ± 0.038 to 0.529 ± 0.023 m). This effect was lost
after the transfer of plants to soil, and from 35 dai onwards
root lengths did not differ significantly in all treatments.
No significant differences were found among treatments and/or time in disease ratings, with values ranging
between 0.75 ± 0.22 and 1.7 ± 0.22. However, mean
lesion length increased significantly (K–W, P < 0.05)
with time in all treatments except for the treatment with isolate Fe10331, in which a reduction in
lesion length occurred from 35 (0.121 ± 0.027 m) to 63
(0.096 ± 0.036 m) dai. Larger differences in lesion length
were observed at 63 dai. At this time, plants inoculated
with either Fe10331 or Fe45121 showed a significant
reduction (K–W, P < 0.05) in the mean lesion length
(0.096 ± 0.036 and 0.097 ± 0.031 m, respectively) compared with control plants (0.289 ± 0.122 m).
Percent effective root length (ERL) did not vary among
treatments and sampling dates, except for Pc123 and Pc21,
which showed a reduction in ERL after transfer to pots
with soil. At 63 dai, ERL of plants with Pc21 (71.1 ± 5.7%)
was significantly lower (K–W, P < 0.05) than that of
the same treatment at the first sampling (96.1 ± 1.4%).
It was, however, statistically similar (K–W, P > 0.05)
to values found for other treatments at the same time
(77.7 ± 4.3 to 97.3 ± 1.7%).
Discussion
Two F. equiseti isolates (Fe10331 and Fe45121) obtained
in Maciá-Vicente et al. (2008a) and screened in vitro for
antagonism against Ggt in Maciá-Vicente et al. (2008b),
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and two isolates of the nematode egg-parasitic fungus
P. chlamydosporia (Pc123 and Pc21), were tested for ability
to colonise barley roots endophytically and for their effects
on plant growth and the take-all caused by Ggt, in pot
trials under greenhouse conditions.
All isolates were able to colonise barley roots, where
they remained endophytically for at least 2 months
under non-axenic conditions. Both F. equiseti and
P. chlamydosporia have been found in previous studies to
colonise barley roots under laboratory conditions using
the same experimental system applied in this work
(Bordallo et al., 2002; Maciá-Vicente et al., 2008b, 2009).
To the best of our knowledge, this is the first longterm study of the endophytic behaviour of both fungal
species under non-axenic conditions. Fusarium equiseti
appeared to be a more efficient rhizosphere coloniser than
P. chlamydosporia, in terms of population size and competition with other fungal root colonisers. Although both
species started with comparable levels of root colonisation
under axenic conditions, both outside and within the root
cortex, transferring the plants to soil affected their capacities to colonise roots in different manners. Fusarium equiseti persisted in the ectorhizosphere at high levels, which
decreased gradually with time, whereas their endophytic
occurrence was constant with slight changes over the
duration of the experiment (approximately 2 months).
The gradual decrease in the presence of F. equiseti in
the ectorhizosphere was accompanied by a corresponding increase in other soil-inhabiting fungi. These fungi
started colonising the ectorhizosphere to a lower extent
than in uninoculated controls. This effect could be related
to the reduction of Ggt growing axenically in culture
tubes observed in Maciá-Vicente et al. (2008b) and could
be an indicator of the competitive ability of the fungus in
the rhizosphere. On the contrary, P. chlamydosporia disappeared rapidly from the root surface after transfer of the
plants to soil, and its frequency in the ectorhizosphere
remained low during the whole experiment. Endophytic
colonisation of P. chlamydosporia remained constant with
time, but was lower than that observed for F. equiseti. In
treatments with P. chlamydosporia, complete colonisation
of the outer root by other soil fungi occurred immediately
after transferring plants to soil, similar to uninoculated
controls. As was observed with F. equiseti, the presence of
P. chlamydosporia reduced the occurrence of other fungi
in the root cortex.
Pochonia chlamydosporia had an early growth-promoting
effect in barley. Both Pc123 and Pc21 increased fresh
shoot and root weight and, at the end of the experiment, the increase reached almost twice that observed
for uninoculated controls. This effect has also been
observed in wheat and tomato plants inoculated with
P. chlamydosporia (Monfort et al., 2005; Siddiqui & Akhtar,
Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors
Journal compilation © 2009 Association of Applied Biologists
J.G. Maciá-Vicente et al.
2008), but in these plants growth promotion was lower
than that found in this study. This is probably because
of our method of fungal inoculation, which was targeted
to plant roots rather than soil. Plant growth promotion
conferred by endophytic colonisation has been described
previously, and this effect can include phenomena such
as direct production of specific substances [i.e. phytohormones such as indole-3-acetic acid (IAA)] (Nassar
et al., 2005), assistance in mineral supply and nutrient
uptake, especially of phosphorus, from soil (Jumpponen
& Trappe, 1998; Sieber, 2002), increase in the availability of carbohydrates and/or CO2 resulting from fungal
metabolism (Jumpponen & Trappe, 1998) or stimulation
of nitrate reduction (Sherameti et al., 2005). Some of
these effects, such as increased phosphorus availability,
are similar to that found for mycorrhizal associations.
In fact, some mutualistic endophytes (i.e. Phialocephala
fortinii) form colonisation patterns similar to the Hartig
net and a thin patchy mantle, typical of ectomycorrhizae
(Fernando & Currah, 1996). The plant growth promotion observed in the present paper does not seem to be
related to formation of rhizospheric hyphal nets. This is
connected with the low root colonisation rates found for
P. chlamydosporia. This points to alternative mechanisms,
such as production by the fungus of specific compounds
(e.g. growth regulators), as the main effectors of plant
growth promotion driven by P. chlamydosporia. Further
studies are required to assess this hypothesis. Re-isolation
methods in culture media used in this study have limitations, which may include a false correlation between the
isolation rate and the real presence of the fungus (Schulz
& Boyle, 2005). It is difficult to discriminate whether
the low levels of P. chlamydosporia found in the roots are
real, or they are an artefact of the methodology applied.
The use of more accurate methods for the detection
and quantification of fungi in the rhizosphere, such as
real-time quantitative PCR (qPCR) techniques or genetic
transformation with the GFP reporter gene in conjugation
with microscopic studies, will help to assess these results
(Ciancio et al., 2005; Rosso et al., 2007; Maciá-Vicente
et al., 2009). In recent research, these methodologies
were developed for the characterisation of the endophytic colonisation of barley roots by both F. equiseti
and P. chlamydosporia under laboratory growth conditions
(Maciá-Vicente et al., 2009). In these studies, first colonisation events consisted of a massive fungal growth on the
root periphery, with single hyphae colonising the cortical
tissues inter- and intracellularly. Subsequent colonisation events involved a reduction of the outer root fungal
biomass and loss of viability of the endophytic mycelium
in some root regions that could be a consequence of elicitation of plant defence responses. In spite of the latter,
well-established viable hyphae were detected in other
Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors
Journal compilation © 2009 Association of Applied Biologists
Root colonisation by endophytes and effects on host plant
root regions, suggesting an evasion of the plant defences
by the fungal endophyte. Both qPCR and confocal laser
scanning microscopy studies with GFP-tagged isolates are
currently being used under the same experimental systems used in the present work to reanalyse these results.
Root behaviour of both F. equiseti and P. chlamydosporia
isolates was also checked in the presence of the cereal
fungal pathogen Ggt. Fe10331 and Pc123 followed
colonisation patterns similar to those observed in
the experiment with the endophytes alone. However,
Fe45121 displayed a reduction in root colonisation rate
and Pc21 showed a higher degree of root colonisation,
than that observed in experiments with the endophytes
alone. After the transfer of barley plants to soil, Ggt
could not be isolated from roots. Ggt rarely produces
asexual structures in culture, and most isolates do not
produce sexual structures at all (Elliott, 2005). Therefore,
differentiation of the dark hyphae of the pathogen
from other pigmented mycelia isolated from barley roots
was difficult or unreliable by means of observation of
morphological characters. Application of a PCR detection
procedure for Ggt (Fouly & Wilkinson, 2000) was
therefore used as an alternative. This method allowed
direct detection of Ggt in the roots of all plants analysed. A
clear suppressive effect by F. equiseti and P. chlamydosporia
on the take-all disease on barley plants could not be
detected, in spite of previous positive results under
laboratory screening conditions (Monfort et al., 2005;
Maciá-Vicente et al., 2008b). Although such preliminary
screening procedures are commonly recommended as
components of a sequence of assays in the search for
biological control agents, quite often results between
simple laboratory and greenhouse practical growing
conditions do not match, as a consequence of the increase
in the experimental variables (Knudsen et al., 1997).
In this study, a technique for inoculation and long-term
root endophytic colonisation by two fungal species was
developed. Non-sterilised sandy soil used in this work
was non-receptive to Pc123 in previous studies (Monfort
et al., 2006). That is, fungal growth of P. chlamydosporia
was inhibited when applied to non-sterilised soil, probably because of soil microbial activities. According to our
results, root tissues of the host plant offer a ‘‘safe’’ environment for the fungus to escape soil competition (‘lack of
receptivity’). Colonisation of the endorhizosphere could
also act as an additional source of fungal inoculum to
the soil. In this sense, previous work reported abundant
chlamydospore production by P. chlamydosporia on the
rhizoplane, and to a lesser extent in the cortex of barley and tomato roots colonised by the fungus (Bordallo
et al., 2002). Independent of their initial root colonisation rates, both types of root endophytes continued to
colonise roots endophytically, although levels were low.
399
Root colonisation by endophytes and effects on host plant
These results suggest that barley roots support certain
root colonisers. It seems that a single inoculation of the
endophytes in culture tubes with barley plants growing
axenically was not enough to keep high endophytic populations of the fungi inoculated. Therefore, their presence
gradually decreased when plants were transferred to soil
to reach a low and stable colonisation level with time.
Long-term endophytic colonisation may hence depend
on an equilibrium between plant defences and the fungal
rhizosphere competence, but could also be influenced by
the methodology used for root inoculation.
Even with the sub-optimal inoculation procedure
of this study, F. equiseti and P. chlamydosporia isolates
persisted as endophytes under non-axenic conditions.
In the case of the putative endophyte P. chlamydosporia,
this resulted in beneficial effects on the host plant growth.
As suggested before, we are currently investigating
practical and theoretical aspects of the root inoculation
of fungi with endophytic capabilities and antagonistic
potential against root pathogens (fungi and nematodes).
The final aim is to optimise their biocontrol performance
and crop growth promotion under agricultural conditions.
Acknowledgements
The authors want to thank Mathieu Gueroult for technical
assistance. This work was supported by grants from
CICYT of the Spanish Ministry of Science and Innovation
(AGL2007-60264 and AGL2008-00716/AGR). J. G. M.
V. was supported by a grant from the Ministerio de
Educación y Ciencia (AP2002-093). L. C. R. and A. C.
acknowledge research funding by Regione Puglia, PE
040, and ELEP, Cornaredo, Italy.
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