Mycorrhiza (2010) 20:191–200

DOI 10.1007/s00572-009-0279-5

ORIGINAL PAPER

Impact of Piriformospora indica on tomato growth

and on interaction with fungal and viral pathogens
Ahmad Fakhro & Diana Rocío Andrade-Linares & Susanne von Bargen &
Martina Bandte & Carmen Büttner & Rita Grosch & Dietmar Schwarz & Philipp Franken

Received: 30 June 2009 / Accepted: 11 September 2009 / Published online: 30 September 2009
# Springer-Verlag 2009

Abstract Piriformospora indica is a root endophytic Keywords Hydroponic cultures . Lycopersicon

fungus with plant-promoting properties in numerous plant esculentum . Pepino mosaic virus . Verticillium dahliae
species and induces resistance against root and shoot
pathogens in barley, wheat, and Arabidopsis. A study over
several years showed that the endophyte P. indica colonised Introduction
the roots of the most consumed vegetable crop tomato. P.
indica improved the growth of tomato resulting in increased Piriformospora indica was originally isolated from the
biomass of leaves by up to 20%. Limitation of disease spore of an arbuscular mycorrhizal (AM) fungus, but
severity caused by Verticillium dahliae by more than 30% inoculation experiments showed its ability to colonise plant
was observed on tomato plants colonised by the endophyte. roots (Verma et al. 1998). It is an anamorphic strain of the
Further experiments were carried out in hydroponic cultures Sebacinales (Basidiomycota), a group with many plant-
which are commonly used for the indoor production of interacting organisms including ecto-, ericoid, and orchid
tomatoes in central Europe. After adaptation of inoculation mycorrhiza (Weiss et al. 2004; Selosse et al. 2007). The
techniques (inoculum density, plant stage), it was shown endophyte P. indica possesses positive influence on growth
that P. indica influences the concentration of Pepino mosaic and development of many different plant species like AM
virus in tomato shoots. The outcome of the interaction fungi. Inoculation leads to increased fresh weights (Varma
seems to be affected by light intensity. Most importantly, et al. 1999), supports the establishment of micro propagated
the endophyte increases tomato fruit biomass in hydroponic plantlets (Sahay and Varma 1999), enhances flower and
culture concerning fresh weight (up to 100%) and dry seed production (Rai et al. 2001; Barazani et al. 2005;
matter content (up to 20%). Hence, P. indica represents a Shahollari et al. 2007), promotes the rooting from cuttings
suitable growth promoting endophyte for tomato which can (Drüge et al. 2007), and results in higher yield (Waller et al.
be applied in production systems of this important 2005). It could be also shown that P. indica induces
vegetable plant not only in soil, but also in hydroponic tolerance against salt stress and resistance against root and
cultures. shoot pathogens (Waller et al. 2005; Serfling et al. 2007;
Deshmukh and Kogel 2007; Sherameti et al. 2008;
A. Fakhro : D. R. Andrade-Linares : R. Grosch : D. Schwarz : Baltruschat et al. 2008; Stein et al. 2008). In contrast to
P. Franken (*) AM fungi however, the endophytic fungus colonises the
Leibniz-Institute for Vegetable and Ornamental Crops, roots and promotes the development of the model plant
Theodor-Echtermeyer-Weg,
Arabidopsis thaliana (Peskan-Berghöfer et al. 2004). In this
14979 Grossbeeren, Germany
e-mail: franken@igzev.de experimental system, a number of plant proteins and genes
were identified which are important for the interaction
A. Fakhro : S. von Bargen : M. Bandte : C. Büttner between the endophyte and the plant root (Shahollari et al.
Faculty of Agriculture and Horticulture, Section Phytomedicine,
2005, 2007; Sherameti et al. 2005, 2008), and the basis for
Humboldt-University Berlin,
Lentzeallee 55/57, induced resistance was analysed (Stein et al. 2008).
14195 Berlin, Germany However, no data are available for the interaction between
192 Mycorrhiza (2010) 20:191–200

P. indica and plants of the Solanaceae which contain al. 2009). There are conflicting reports on yield losses due
important crop species and models like potato, tomato, to PepMV infection varying from low up to the collapse of
tobacco, eggplant, or petunia. the crop (Soler-Aleixandre et al. 2005). Apart from total
Tomato is the most commonly grown fresh market yield losses, significant decreases in fruit quality, and thus,
vegetable worldwide, but various pathogens can lead to marketable yield reductions up to 40% are reported (Spence
dramatic losses in yield. One widespread disease of field et al. 2006; Schwarz et al. 2009).
grown tomato plants is verticillium wilt caused by the The present investigation was carried out first to analyse,
soilborne fungus Verticillium dahliae (Pegg and Brady if the endophyte P. indica is able to reduce the symptoms of
2002). The disease symptoms are characterised by V- verticillium wilt in substrate-grown tomato. Secondly, it
shaped yellowing of the leaves, browning of vascular was aimed to establish the interaction between P. indica and
bundles, and wilting. The fungus infects the root system tomato in hydroponic culture systems for analysing, if the
directly or through wounds, invades the xylem, and moves accumulation of PepMV within the apical shoot is
upward. Once in plant tissues, it produces toxins (Mansoori influenced by fungal colonisation of the roots. At third,
et al. 1995), and in course of the interaction, the disease the impact of P. indica on tomato fruit biomass in the
symptoms progress up the stem, and the plant becomes hydroponic system was determined.
stunted (Gold et al. 1996). Control strategies are soil
fumigation, crop rotation, and growing cultivars with the
Ve resistance gene (Talboys 1984; Huisman and Ashworth Materials and methods
1976; Ligoxigakis and Vakalounakis 1994). However, these
strategies are limited by the longevity of the V. dahliae Cultivation of fungi and Pepino mosaic virus
microsclerotia in soil (Pegg 1974; Talboys 1984), by the
economically unpractical long rotation cycles (Harrington The endophyte P. indica was propagated on potato dextrose
and Dobinson 2000) and by the appearance of new race 2 agar (PDA, VWR, Berlin, Germany) at 28°C. Chlamydo-
strains which cause typical disease symptoms also on spores were collected after 2 weeks from the agar plate and
resistant Ve cultivars (Dobinson et al. 1996) used to inoculate 100 ml potato dextrose broth (PDB,
Besides production in the field, the importance of VWR) in 300-ml Erlenmeyer flasks. These flasks were
soilless cultivation of tomato using open or closed further incubated at 28°C and 90 rpm for 4 weeks.
hydroponic systems has been increasing worldwide during Mycelium and spores were filtered through gauze and
the last three decades (Savvas 2003). In these production mixed with a blender (Model D72, Moulinex, Leipzig,
systems, plants are usually supplied with a nutrient solution Germany) for 2 min at lowest speed in 100 ml of sterile tap
circulating to allow a more accurate control of the root water. Propagule (chlamydospores, hyphal fragments) con-
environment. This can result in an optimal use of water and centration was examined in a Thoma chamber and the
nutrients, and thus, in higher yields and better fruit quality. number of viable propagules by plating on PDA.
However, recirculation of the nutrient solution and high V. dahliae (kindly provided by Valerie Grimault,
plant density facilitates the rapid and efficient spread of GEVES, Angers, France) was grown in 150-ml sucrose
pathogens and may, thus, increase the risk for epidemics if sodium nitrate liquid medium (Sinha and Wood 1968) at
not managed well (Stanghellini and Rasmussen 1994). 28°C and 100 rpm. After 1 week, the culture was
Since 1999, Pepino mosaic virus (PepMV) has attracted transferred to 200-ml fresh medium and further incubated
much attention because it is found widely in tomato for 2 weeks. The culture was mixed by a blender (Model
greenhouses in many European countries, in Morocco, D72, Moulinex) for 40 s at minimal speed. The mixed
South and North America, and in China (see references in solution was filtrated and washed once by centrifugation.
Spence et al. 2006). The origin of its sudden occurrence is The number of conidia was estimated by counting in a
not clear. However, rapid transmission and spread of Thoma chamber, and their viability was checked by plating
PepMV within and between greenhouses can be mechan- on PDA. For inoculation, the suspensions were adjusted
ically caused by tools, clothes, and the hands of workers with sterile tap water to a concentration of 105 conidia per
during crop handling (Jones et al. 1980). Typical symptoms millilitre based on counted colonies.
of infected leaves are rolling, light yellow mosaics, dark The virus isolate Pepino mosaic virus-Sav E397 used
green discolouration, and leaf distortion (Jordá et al. 2001; in all experiments was obtained from tomatoes purchased
Van der Vlugt et al. 2002). Fruits may show yellow in a German supermarket that were labelled to be
blotches, necrotic or yellow spots, and irregular ripening imported from France. The virus isolate was recovered
(French et al. 2008). Symptom expression can be affected from these tomato fruits by maceration of crude fruit in
by the tomato cultivar, the genotype of the virus, and the ELISA sample buffer (10% phosphate-buffered serum
environmental conditions (French et al. 2008; Hanssen et (PBS) buffer, 2% polyvinylpyrrolidone) followed by
Mycorrhiza (2010) 20:191–200 193

mechanical inoculation using 0.05% Celite as an abrasive was determined 1 week later by double antibody sandwich
to tobacco plants (Nicotiana benthamiana) on which it (DAS)-ELISA (see below).
was further propagated as described (Schwarz et al. 2009).
Analysis of plants
Cultivation and inoculation of tomato plants
Disease severity in V. dahliae-inoculated plants was
For the experiment with the pathogen V. dahliae, surface assessed at harvest (8 weeks after sowing, 6 weeks after
disinfected tomato seeds (Lycopersicon esculentum Mill. P. indica inoculation, and 4 weeks after pathogen inocula-
cv. Hildares) were germinated on 0.8% water agar for tion) based on an arbitrary scale of disease classes: 0=no
1 week and subsequently transplanted into pots containing symptoms, 1=slight yellowing of leaf, stunting, or wilting,
1 l of the substrate “Fruhstorfer Erde Typ P” (Archut, 2=moderate yellowing of leaf, stunting, or wilting, 3=severe
Vechta, Germany). These pot cultures were placed for the yellowing of leaf, stunting, or wilting, and 4=leaf death.
whole experiment in a greenhouse at day/night temperature Values were estimated for the different set of plants using
of 25°C/19°C, relative humidity 54%/69%, and a mean the formula ðΣ n
of leavesdisease class  disease classÞ =
daily radiation of 362 Mol m−2 day−1. The plants were total number of leaves. The value obtained for the control
watered twice per week with 40 ml of nutrient solution (De plants infected with V. dahliae was set at 100%. In parallel,
Kreij et al. 1997; pH=5.5; electric conductivity (EC)= pieces of the stem base were placed on PDA, and the
2 dS m−1). Half of the plants (36) were inoculated with outgrowth was microscopically confirmed. Fresh and dry
P. indica before planting into “Fruhstorfer Erde Type P” by weights of shoots were estimated at harvest time.
dipping the roots overnight in a suspension of 105 cfu/ml of PepMV infection of tomato plants was determined by
tap water. In addition, fresh P. indica mycelium was mixed testing the upper most leaves in DAS-ELISA (modified after
1/100 (w/w) with this substrate to achieve heavily colonised Clark and Adams 1977) using commercially available
plants. Two weeks after inoculation with P. indica, half of polyclonal antibodies (immunoglobulin IgG) according to
the plants (18 colonised by P. indica and 18 controls) were the instructions provided (AS-0632; DSMZ, Braunschweig,
drenched with 30 ml of the conidia suspension of V. dahlia Germany). Each ELISA test included a negative and a
(105 conidia per millilitre). positive control. Samples were rated positive if the absor-
For all other experiments (Table 1), seeds were germi- bance measured at 405 nm was greater than twice the level
nated for 2 weeks in sterilised sand and further grown in obtained from healthy controls (Cordoba-Selles et al. 2007).
pots filled with 0.5 l sterilised sand (v/v: 1 (0.2–1 mm)/ Fresh and dry weights of shoots were measured at the
1 (2–3 mm); Euroquarz, Laußnitz, Germany) fertilised with end of all experiments, while fresh and dry weights of fruits
the nutrient solution mentioned above. One to 4 weeks at the end of experiments 4 and 5 (Tab. 1). Dry matter
later, plants were inoculated by dipping the roots for 2 h in content was calculated.
a P. indica suspension or a control solution (Table 1).
Thereafter, inoculated plants and controls were transferred Statistical analysis
to buckets or gullies (ten plants per biological replicate),
containing the nutrient solution, sand (as above), or Disease severity after V. dahliae infection was analysed by
substrate (Fruhstorfer Erde Type P). Twice a week, nutrient the nonparametric Kruskal–Wallis test, while all other
solution was renewed or added to sand or substrate until it experimental data were processed by analysis of variance
drained off the buckets. In gullies, nutrient solution was procedures. Means at the different measurement dates were
applied as described (De Kreij et al. 1997). Daily climate separated by Tukey’s test procedure at P=0.05. Significant
data averages are shown in Table 1. For ensuring root differences are presented by different letters, standard
colonisation by P. indica, five to six fragments (3–4 cm) of deviation bars are added in the figures, and significant
plant roots were sampled 10 days after inoculation, interactions between factors are mentioned in the legends.
incubated on PDA, and analysed for the appearance of STATISTICA 6.0 software (2003) was used for all
chlamydospores with characteristic morphology (Verma et statistical analyses indicated in the figure legends.
al. 1998) by means of light microscopy. Two weeks after
inoculation with P. indica, when tomato plants had developed
nine to ten leaves, half of the P. indica-inoculated or the Results
control plants were mechanically inoculated on leaf number
8 or 9 by abrading a homogenate containing PepMV which Impact of P. indica on verticillium wilt
was obtained from leaves of host plants (N. benthamiana)
using 0.05% Celite as an abrasive. These leaves were, Tomato plants grown in substrate and infected with the
thereafter, washed with sterile water. Successful infection pathogen V. dahliae showed typical symptoms as leaf
194 Mycorrhiza (2010) 20:191–200

Table 1 Conditions of tomato growth in soilless cultures

Experiment number 1 2 3 4 5a 5b

Date of P. indica 3–6; 102–110 4; 104–105 4; 104–105 4; 104–105 4; 104–105 4; 104–105

inoculation (weeks
after sowing;
developmental stagea)
Inoculum density 3 3 or 9 3 3 3 3
(105 cfu/ml)
Substrate Sand Sand, substrate, Nutrient Nutrient Nutrient Nutrient
nutrient solution solution solution solution
solution
Containment 1 l buckets 2 l buckets Gullies Gullies 10 l buckets 10 l buckets
Pathogen No No PepMV PepMV PepMV PepMV
Plants per treatment 6 6 2×10 4×10 4 4
Temperature 21.0/17.6 22.2/19.2 21.7/20.0 23.4/18.9 25.8/20.8 25.8/20.8
(day/night; °C)
Humidity 63.2/63.8 59.1/57.4 78.2/75.3 76.3/79.9 67.4/78.8 67.4/78.8
(day/night; %)
Mean daily radiation 18.5 7.8 9.0 12.8 11.0 6.0
(Mol m−2 d−1)
CO2 concentration 479.6 454.5 365.7 396.3 452.0 452.0
(ppm)
End of the experiment 9 11 10 13 12 12
(weeks after sowing)
a
According to Feller et al. (1995)

yellowing, stunted growth, wilting, and death of the plant. 1995). This experiment showed in addition that the medium
Fresh weights were significantly reduced in the pathogen- used for growing P. indica had no influence on tomato
infected control plants, while the dry matter content was biomass as it has been seen before in soil cultures (Varma et
only slightly different (Fig. 1a). Tomato plants inoculated al. 1999).
with the endophyte P. indica showed a significant The second experiment (Table 1) was carried out for
increase in fresh weights. Moreover, the negative effect analysing the effects of growth medium and inoculum
of the pathogen on plant growth was alleviated when the density on tomato growth (Fig. 3). Comparison of substrate,
plants were colonised by the root endophytic fungus. sand, and nutrient solution showed a significant positive
Differences in fresh weight and dry matter content effect of P. indica on shoot fresh weight with a low density
between P. indica-inoculated plants and the corresponding of inoculum only in sand. Significant negative effects were
controls were higher, when the plants were infected with obvious with a high inoculum dosage in nutrient solution.
V. dahliae. This was mirrored by the result of a two-way No differences were detected for the dry matter content and
analysis of variance (ANOVA) showing a significant interaction between the two factors “inoculum density” and
interaction between the two factors “pathogen” and “growth medium” could not be observed.
“endophyte.” Estimation of the disease symptoms also
showed the protecting effect of P. indica (Fig. 1b). The Impact of P. indica on Pepino mosaic virus spread
disease severity was reduced by 32% in plants colonised
by the endophytic fungus. Experiments 3–5 (Table 1) were carried out to analyse the
influence of P. indica on the concentration of PepMV.
Establishment of P. indica inoculation in a soilless system Typical symptoms of PepMV infection were observed in all
experiments, as it has been described by Jordá et al. (2001)
In order to reveal the best conditions for P. indica infection and Van der Vlugt et al. (2002), but not quantified. The
in soilless systems, at first, the optimal plant stage of concentration of PepMV decreased over time (14, 41, and
inoculating the endophyte was determined (experiment 1 in 57 days after inoculation (dai)) in the upper most leaves in
Table 1). For this purpose, tomato plants were inoculated at experiment 3, but was always between 10% and 20%
different stages (Fig. 2). Significant positive effects were higher in tomato plants colonised by P. indica than in
only detected when the fungal inoculum was added 4 weeks noncolonised controls (Fig. 4a). The difference was
after sowing (stage 104–105 according to Feller et al. significant at the latest date (57 dai). The virus responded
Mycorrhiza (2010) 20:191–200 195

100
(a) a two dates (17 and 31 dai). In plants however, which obtained
b - P. indica
shoot fw [g/plant]
80 + P. indica b higher light intensities, a decreased virus concentration was
c detected in the leaves at the last two dates (31 and 59 dai)
60 when the roots were colonised by the endophytic fungus
(significant at 59 days after inoculation).
40

20 Impact of P. indica on fruit biomass

0 Higher numbers of flowers or setting of fruits were

ab a
12 bc c observed in the plants inoculated with P. indica in all
experiments. Plants of experiment 4 and 5 were, therefore,
shoot dmc [%]

9 used to harvest and to analyse the fruits (Fig. 5 shows

results of experiment 5). This revealed a significant
6
influence of P. indica on total fruit biomass. (Yield of
3 marketable fruits was not determined). At the date of
harvest, tomato fruit fresh weights per plant were increased
0 between 50% and 100% and dry matter content between
- V. dahliae + V. dahliae
120 10% and 20%. The increase in fresh weights was not due to
(b) differences in the single fruit, but due to higher numbers of
disease severity (%)

100
fruits. Significant differences were also obtained in experi-
80 ment 4 with fresh weight increases between 40% and 50% and
60 a 7% higher dry matter content (data not shown).

40

20 Discussion
0
control P. indica P. indica is a root-endophytic fungus with plant growth-
promoting abilities. The increase of fresh weights was in
Fig. 1 Influence of P. indica on tomato–V. dahliae interaction. Tomato
plantlets were transferred to pots containing substrate supplemented or some studies between 20% and 40% (Peskan-Berghöfer et
not with P. indica. After 2 weeks, half of the controls and the inoculated al. 2004; Barazani et al. 2005) while it could reach in others
plants were infected or not with the pathogen V. dahliae. Four weeks up to 100% (Varma et al. 1999; Waller et al. 2005; Serfling
later, shoot fresh weights (fw) and dry weights were measured, and dry et al. 2007). This characteristic could be confirmed in the
matter content (dmc) was calculated (a). Significant different values are
indicated by different letters above the columns. The factors P. indica present experiments using tomato as a plant host. Fresh
and V. dahliae showed a significant interaction for both parameters weight of P. indica-colonised plants were, however, in the
(two-way ANOVA; P=0.05; n=18). In addition, disease severity was best case not more than 20% higher than in controls and
estimated and set for the control plants as 100% (b). Statistical analysis reached although significant in most experiments increases
showed that disease severity was significantly different (Kruskal–Wallis
test; P=0.05; plants) of only 10%. This is probably on the one hand dependent
on the plant species. For instance, rooting of cuttings was
strongly enhanced in Euphorbia pulcherrima and Pelargo-
opposed in experiment 4 (Fig. 4b). First, virus concentra- nium x hortorum, while no effect could be observed in
tion increased during the course of the experiment (10, 38, Petunia hybrida although cultivated under the same
64, and 81 dai). Secondly, the virus was detected at all dates conditions (Drüge et al. 2007). On the other hand,
except the second (38 dai) with lower concentrations in conditions of inoculation (plant stage, substrate, and
plants, which were inoculated with the root endophyte, than inoculum density) and growth clearly played an important
in the controls. This reduction of virus spread was role. Best results for tomato were obtained in sand
significant at the first date of sampling (10 dai). In order compared to substrate and nutrient solution. A similar
to find out the differences between the two experiments, result comparing sand and soil has been obtained with
climate conditions during the cultivation were compared, wheat (Serfling et al. 2007). Nutrient poor conditions in the
and light intensity was revealed as the major variation sand compared to the substrate cannot be the reason, since
between the two experiments (Table 1). Consequently, half nutrient supply was optimal, and P. indica does not improve
of the plants were shaded in experiment 5 (Fig. 4c). In these at least P and N content in tobacco (Barazani et al. 2005) or
shaded plants, P. indica inoculation led to a significantly in barley (Achatz et al. unpublished). Hence, an explanation
increased content of PepMV in the apical leaves at the first for the influence of the substrate has for the moment to be
196 Mycorrhiza (2010) 20:191–200

300 a
b b b b b

shoot fw [g/plant]
250
200
150
100
50
0
a ab
c bc bc bc
10
8
shoot dmc [%]

6
4
2
0
control culture 3w 4w 5w 6w
medium

Fig. 2 Influence of inoculation date. Tomato plantlets (3 weeks after fungus. Nine weeks after sowing, shoot fresh weights (fw) and dry
sowing) were transferred to pots containing a nutrient solution and weights were measured, and dry matter content (dmc) was calculated.
supplemented with the P. indica inoculum immediately (3 weeks after Significant differences between different types of inoculum are
sowing) or after 7, 14, or 21 days (4–6 weeks after sowing). Control indicated by different letters above the columns (one-way ANOVA;
plants obtained no supplement (control) or culture medium without the P=0.05; n=6)

left open. The other conditions tested for tomato were the
inoculum density and the plant stage of inoculation. This
showed that the amount of fungus should not exceed a
inoculum density
certain extent, because negative effects on plant growth
300 none were obtained. Such negative effects with high amounts of
3 x 105 cfu/ml
250 9 x 105 cfu/ml inoculum of P. indica have been also seen in an experiment
shoot fw [g/plant]

with tomato using field soil as substrate (data not shown).

200 Another study using poplar as host in a Petri dish system
150 also revealed negative effects of the endophyte (Kaldorf et
al. 2005). This was not dependent on inoculum amount, but
100
on the mode of cultivation of the fungus. If P. indica was
50 grown on media containing ammonium as N source, the
0
fungus started to colonise not only the cortex, but also the
vascular cylinder of the roots, and necrotic lesions
10 occurred. Positive effects were observed, if the endophyte
shoot dmc [%]

8 derived from cultures with nitrate. Negative effects could be

based on the mode of colonisation. The fungus is not a
6
biotroph as e.g., the arbuscular mycorrhizal fungi, but
4 increases the number of dead cells in the root (Franken et
al. 2000). Interestingly, P. indica seems to induce the
2
programmed cell death of plants as numerous pathogens do,
0
nutrient solution sand substrate
but in contrast to these pathogens, the colonisation of the
root by the endophyte depends on the cell death programme
Fig. 3 Influence of cultivation system and P. indica inoculum density. of the plant (Deshmukh et al. 2006). If the number of dead
Tomato plantlets were transferred into buckets containing a nutrient
solution, sand, or a commercial garden substrate. Each cultivation
cells in the root exceeds a particular threshold, P. indica
system was supplemented with no, with 3, or with 9×105 cfu/ml of P. could exert a negative influence on plant growth and
indica inoculum. Shoot fresh weights (fw) and dry weights were development. This might be the case, if the amount of
measured, and dry matter content (dmc) was calculated 8 weeks after inoculum is too high and the colonisation is from the
inoculation. Two-way ANOVA (P=0.05; n=6) revealed significant
differences in shoot fw for the influence of inoculum density and of
beginning too intense. An optimal balance between positive
the cultivation system, but not for the interaction of the two factors. and negative effects of P. indica could also explain the
No significant differences were revealed for shoot dmc observation that best effects were obtained, if the fungus
Mycorrhiza (2010) 20:191–200 197

1.4 700
(a) low light, - P. i. high light, - P. i.
- P. i.
1.2 + P. i. 600 low light, + P. i. high light, + P. i.

fruit fresh weight [g/plant]

adsorbance at 405 nm

1.0 500
0.8 *
400
0.6
300
0.4
200
0.2
100
0
14 dai 41 dai 57 dai
0
0.7
(b) 9
- P. i.
adsorbance at 405 nm

0.6

fruit dry matter content [%]

+ P. i. 8
0.5 7
0.4 6
0.3 5
0.2 4
* 3
0.1
2
0
10 dai 38 dai 64 dai 81 dai 1
0.5 0
(c) low light, - P. i. high light, - P. i. without PepMV with PepMV
low light, + P. i. high light, + P. i.
adsorbance at 405 nm

0.4
Fig. 5 Influence of P. indica on fruit fresh weight and dry matter
* content. Tomato plants were grown in nutrient solution under two
0.3 * light regimes and inoculated or not with P. indica and Pepino mosaic
*
virus. Twelve weeks after germination, fruits were harvested, fresh
0.2 and dry weights measured, and dry matter content calculated. A three-
way ANOVA (P=0.05; n=4) showed significant influence on fresh
0.1 weight for P. indica and on dry weight for all three factors (light,
PepMV, and P. indica). Interactions between any of the factors were
0 not detected
17 dai 31 dai 59 dai

Fig. 4 Influence of P. indica on Pepino mosaic virus spread. Tomato and future experiments will be directed to proof more
plants were grown in nutrient solution in three consecutive years and variables in this respect. Preliminary experiments indicate
inoculated or not with P. indica. a Winter 2006. b Summer 2007.
for instance that the culture medium for growing the
c Late summer 2008 under two light regimes When roots were
colonised, youngest leaves of half of the plants were inoculated with fungus also seems to influence the effect of the
PepMV. At different days after inoculation (dai), youngest leaves were endophyte on plant performance. In addition, different
harvested, and PepMV colonisation was measured by ELISA. isolates of Sebacina vermifera, a close relative of P.
Significant differences between plants inoculated or not by P. indica
indica, have to be tested, if they promote tomato growth
are indicated by asterisks. (One-way (a, b) and two-way (c) ANOVA;
P=0.05; n=2 (a) or 4 (b, c)). An interaction between light and P. even more as it has been shown for tobacco and barley
indica was detected at 31 dai (c) (Barazani et al. 2005; Deshmukh et al. 2006). The
mechanisms behind the growth-promoting effects of P.
indica are a matter of debate. Phytohormones as ethylene,
was applied at the four to five leaf stages. At the earlier auxin, and cytokinin seem to play a role as different
date, the percentage of dying cells could reach the point, analyses of the fungal culture filtrate and particular plant
where positive and negative effects are in equilibrium, mutants indicate (Barazani et al. 2007; Sirrenberg et al.
while at the later dates, the root is not susceptible anymore 2007; Vadassery et al. 2008). To make things even more
for the positive activity of the fungus. Variable effects of P. complicated, a recent analysis has shown that P. indica
indica on vegetative growth are probably not simply due to contains bacteria inside its cytoplasm which show similar
the extent of colonisation, because plants showing between effects on plant growth and defence reactions as the
10% and 50% colonisation intensities were not different in fungus (Sharma et al. 2008).
their shoot fresh weights (data not shown). All this In addition to promoting vegetative growth, P. indica
indicates that the outcome of the interaction between also exerts a positive influence on the generative organs of
tomato and P. indica depends on experimental conditions, plants. In contrast to the relatively low enhancement of
198 Mycorrhiza (2010) 20:191–200

shoot fresh weights, the increase in tomato fruit biomass pathogen in the plant. Such resistance reactions induced by
was surprisingly high. This was not due to increased P. indica have been observed in case of powdery mildew in
fresh weight of single fruits, but the fruit number was barley, wheat, and Arabidopsis (Waller et al. 2005; Serfling
larger than in control plants. Higher number of inflor- et al. 2007; Stein et al. 2008). The increased production of
escences and fruit settings was already observed in the antioxidants in roots and shoots of P. indica-colonised
experiments 1–4 (data not shown). In addition to the plants were discussed as one reason for the induced
fresh weight, P. indica also enhanced the dry matter resistance (Waller et al. 2005; Serfling et al. 2007), and
content of the fruits. This indicates that more biomass was also, particular genes known to be involved in plant defence
transported into the generative organs during the growth reactions were shown to be systemically induced after P.
period of the plants. Because the vegetative organs were indica inoculation (Waller et al. 2008). Such type of studies
not reduced in size, there must have been a higher could be also carried out for tomato, where many defence
production of these biomasses during growth of the and pathogenesis-related genes are known.
tomatoes in interaction with the endophyte. In barley, Although not significant at all dates of investigation,
were yield increases up to 11% were observed in open the overall picture of the last three experiments sug-
door experiments (Waller et al. 2005), different parameters gested that P. indica interferes with Pepino mosaic virus
have been tested (Achatz et al. submitted). While accumulation in the apical shoot and that the outcome of
improved mineral nutrition or protection against patho- this interference is dependent on light intensities during
gens did not play a role, enhanced CO2 assimilation under tomato cultivation. Such interactions between fungal root
low light conditions was observed. Indeed, in the last endophytes and viral pathogens were up to now only
experiment without the virus, fresh weights were in- reported for arbuscular mycorrhizal fungi and the tobacco
creased by a factor of 2.3 under low light and by a factor mosaic virus (Dehne 1982; Shaul et al. 1999). In both
of 1.9 under high light. This difference was not significant cases, viral occurrence and resulting symptoms were
as no interaction could be observed between the two increased in leaves. In contrast, when tomatoes were
factors endophyte and light. Nevertheless, further experi- coinoculated with PepMV and a fungal pathogen, such as
ments will be carried out with higher differences in the light Verticillium spp. (Spence et al. 2006) or Pythium aphani-
intensities and measurements of C assimilation and total C in dermatum (Schwarz et al. 2009), the virus colonisation of
the different organs of the tomato plant in order to better the plant was inhibited. The root necrosis caused by the
elucidate the basis for the increased biomasses. In addition, the fungal pathogens could perhaps induce resistance mecha-
total yield until the last fruit setting has to be monitored for nisms affecting virus multiplication and spread (Van Loon
excluding the possibility that P. indica-colonised plants are 1997), and/or biochemical and structural changes in root
just developmentally progressed compared to controls. Such architecture reduced the efficiency of PepMV uptake of
an accelerated development was indicated by particular gene roots through the nutrient solution. Similar to the AM
expression patterns in barley roots (Waller et al. 2008). fungi, P. indica might increase under low light conditions
However, yield of seeds in tobacco, barley, and Arabidopsis the carbohydrate content of cells due to a higher
was not only increased at a particular date, but also overall at photosynthetic rate and in this way, stimulates the number
the end of the whole growth period (Barazani et al. 2005; of virus particles in the tissues as detected by ELISA.
Waller et al. 2005; Shahollari et al. 2007). Increases in C assimilation of P. indica-colonised plants
In barley, in wheat, and in Arabidopsis, it has been have been observed in barley (Achatz et al. submitted) and
shown that P. indica is able to alleviate the symptoms after in P. x hortorum (unpublished). The difference in
attack of the plants by fungal pathogens (Waller et al. 2005; carbohydrate contents between control and endophyte-
Serfling et al. 2007; Stein et al. 2008). It was, therefore, not colonised plants would be lower under high light
surprising to find that the endophyte is also reducing the conditions, and another mechanism would become evi-
symptoms of verticillium wilt in tomato. The disease dent. Such a mechanism could be similar to the systemic
severity was lowered by more than 30%, and P. indica induced resistance (SIR) against viruses which was
balanced the fresh weight decrease caused by the pathogen. obtained with particular plant growth-promoting rhizobac-
This was in the range what has been observed in the other teria (Raupach et al. 1996; Jetiyanon and Kloepper 2002)
systems mentioned in the introduction. A similar effect on and results in a decrease of the accumulation of PepMV in
verticillium wilt has been also observed by using the tomato. It has to be mentioned that usually systemic
nonvirulent isolate Dvd-E6 of V. dahliae as a competitor acquired resistance and not SIR is acting against viruses
(Chen et al. 2004). In this case, the colonisation of Dvd-E6 (Ton et al. 2002). However, an SIR-similar mechanism
nearly totally excluded the infection by the virulent race dependent on jasmonate signalling was revealed as being
(Shittu et al. 2009). If this is also the case for P. indica responsible for the reduction of powdery mildew in P.
remains to be analysed by assessing the biomass of the indica-colonised Arabidopsis plants (Stein et al. 2008).
Mycorrhiza (2010) 20:191–200 199

Conclusion Drüge U, Baltruschat H, Franken P (2007) Piriformospora indica

promotes adventitious root formation in cuttings. Sci Hortic
112:422–426
P. indica reduces the disease symptoms caused by the Feller C, Bleiholder H, Buhr L, Hack H, Hess M, Klose R, Meier U,
fungal pathogen V. dahliae and is able to repress the Stauss R, van den Boom T, Weber E (1995) Phänologische
amount of Pepino mosaic virus provided that light Entwicklungsstadien von Gemüsepflanzen: II. Fruchtgemüse und
intensities are high. Tomato plants colonised by the Hülsenfrüchte. Nachr bl Dtsch Pflanzenschutzd 47:217–232
Franken P, Requena N, Bütehorn B, Krajinski F, Kuhn G, Lapopin L,
endophyte show only slightly enhanced vegetative develop- Mann P, Rhody D, Stommel M (2000) Molecular analysis of the
ment, but fruit biomass is strongly increased. More research is arbuscular mycorrhiza symbiosis. Arch Agron Soil Sci 45:271–
necessary to further optimise the application of P. indica and 286
to ensure that quality of fruits concerning taste- and health- French CJ, Dubeau C, Bunckle A, Ferguson G, Haesevoets R,
Bouthillier M, Bernardy MG (2008) Overview of Pepino mosaic
related compounds are not negatively affected. The presented virus research. Can J Plant Pathol 30:373–374
results, however, let us already suppose that the plant- Gold J, Lee B, Robb J (1996) Colonization of tomatoes by Verticillium
protecting and development-promoting abilities of P. indica dahliae: determinative phase II. Can J Bot 74:1279–1288
could be used to improve the production of tomatoes in Hanssen IM, Paeleman A, Vandewoestijne E, Van Bergen L, Bragard
C, Lievens B, Vanachter ACRC, Thomma BPHJ (2009) Pepino
hydroponic cultures. mosaic virus isolates and differential symptomatology in tomato.
Plant Pathol 58:450–460
Acknowledgements A. Fakhro is supported by a scholarship from Harrington MA, Dobinson KF (2000) Influences of cropping practices
Al-Furat University (Syria) and D. R. Andrade-Linares by a grant of on Verticillium dahliae populations in commercial processing
the German Academic Exchange Service. tomato fields in Ontario. Phytopathology 90:1017
Huisman OC, Ashworth LJ Jr (1976) Influence of crop rotation on
survival of Verticillium albo-atrum in soils. Phytopathology
66:978–981
References Jetiyanon K, Kloepper JW (2002) Mixtures of plant growth-promoting
rhizobacteria for induction of systemic resistance against multiple
Baltruschat H, Fodor J, Harrach BD, Niemczyk E, Barna B, Gullner plant diseases. Biol Control 24:285–291
G, Janeczko A, Kogel KH, Schäfer P, Schwarczinger I, Zuccaro Jones RAC, Koenig R, Lesemann DE (1980) Pepino mosaic virus, a
A, Skoczowski A (2008) Salt tolerance of barley induced by the new potexvirus from pepino (Solanum muricatum). Ann Appl
root endophyte Piriformospora indica is associated with a strong Biol 94:61–68
increase in antioxidants. New Phytol 180:501–510 Jordá C, Lázaro Pérez A, Martínez-Culebras PV (2001) First report of
Barazani O, Benderoth M, Groten K, Kuhlemeier C, Baldwin IT Pepino mosaic virus on natural hosts. Plant Dis Notes 85:1292
(2005) Piriformospora indica and Sebacina vermifera increase Kaldorf M, Koch B, Rexer KH, Kost G, Varma A (2005) Patterns of
growth performance at the expense of herbivore resistance in interaction between Populus Esch5 and Piriformospora indica: a
Nicotiana attenuata. Oecologia 146:234–243 transition from mutualism to antagonism. Plant Biol 7:210–218
Barazani O, Von Dahl CC, Baldwin IT (2007) Sebacina vermifera Ligoxigakis EK, Vakalounakis DJ (1994) The incidence and
promotes the growth and fitness of Nicotiana attenuata by distribution of races of Verticillium dahliae in Crete. Plant
inhibiting ethylene signaling. Plant Physiol 144:1223–1232 Pathol 43:755–758
Chen P, Lee B, Robb J (2004) Tolerance to a non-host isolate of Mansoori B, Milton JM, Smith CJ (1995) Isolation and partial
Verticillium dahliae in tomato. Physiol Mol Plant Pathol 64:283– purification of a phytotoxin related to pathogenic Verticillium
291 species. J Phytopathol 143:33–36
Clark MF, Adams AN (1977) Characteristics of microplate method of Pegg GF (1974) Verticillium diseases. Rev Plant Pathol 53:157–182
enzyme-linked immunosorbent assay for detection of plant Pegg GF, Brady BL (2002) Verticillium wilts. Oxford, UK
viruses. J Gen Virol 34:475–483 Peskan-Berghöfer T, Shahollari B, Giong PH, Hehl S, Markert C,
Cordoba-Selles MC, García-Rández A, Alfaro-Fernández A, Jordá- Blanke V, Kost G, Varma A, Oelmuller R (2004) Association of
Gutiérrez C (2007) Seed transmission of Pepino mosaic virus Piriformospora indica with Arabidopsis thaliana roots represents
and efficacy of tomato seed disinfection treatments. Plant Dis a novel system to study beneficial plant-microbe interactions and
91:1250–1254 involves early plant protein modifications in the endoplasmic
De Kreij C, Voogt W, van den Bos AL, Baas R (1997) Voedingso- reticulum and at the plasma membrane. Physiol Plant 122:465–
plossingen voor de teelt van tomaat in gesloten teeltsystemen. 477
Brochure VG Tomaat, The Netherlands Rai M, Acharya D, Singh A, Varma A (2001) Positive growth
Dehne HW (1982) Interaction between vesicular-arbuscular mycorrhizal responses of the medicinal plants Spilanthes calva and Withania
fungi and plant pathogens. Phytopathology 72:1115–1119 somnifera to inoculation by Piriformospora indica in a field trial.
Deshmukh SD, Kogel KH (2007) Piriformospora indica protects Mycorrhiza 11:123–128
barley from root rot caused by Fusarium graminearum. J Plant Raupach GS, Liu L, Murphy JF, Tuzun S, Kloepper JW (1996)
Dis Prot 114:263–268 Induced systemic resistance in cucumber and tomato against
Deshmukh SD, Hueckelhoven R, Schaefer P, Imani J, Sharma M, Cucumber mosaic cucumovirus using plant growth-promoting
Weiss M, Waller F, Kogel KH (2006) The root endophytic fungus rhizobacteria (PGPR). Plant Dis 80:891–894
Piriformospora indica requires host cell death for proliferation Sahay N, Varma A (1999) Piriformospora indica, a new biological
during mutualistic symbiosis with barley. Proc Natl Acad Sci hardening tool for micropropagated plants. FEMS Letters
USA 103:18450–18457 181:297–302
Dobinson KF, Tenuta GK, Lazarovits G (1996) Occurrence of race 2 Savvas D (2003) Hydroponics: a modern technology supporting the
of Verticillium dahliae in processing tomato fields in southwestern application of integrated crop management in greenhouse. J Food
Ontario. Can J Plant Pathol 18:55–58 Agric Environ 1:80–86
200 Mycorrhiza (2010) 20:191–200

Schwarz D, Beuch U, Fakhro A, Bandte M, Büttner C, Obermeier C Soler-Aleixandre S, Lopez C, Diez MJ, de Castro AP, Nuez F (2005)
(2009) Spread and interaction of Pepino mosaic virus (PepMV) Association of Pepino mosaic virus with tomato collapse. J
and Pythium aphanidermatum in a closed nutrient solution Phytopathol 153:464–469
recirculation system: effects on tomato growth and yield. Plant Spence NJ, Basham J, Mumford RA, Hayman G, Edmondson R,
Pathol, accepted Jones DR (2006) Effect of Pepino mosaic virus on the yield and
Selosse MA, Setaro S, Glatard F, Richard F, Urcelay C, Weiss M quality of glasshouse-grown tomatoes in the UK. Plant Pathol
(2007) Sebacinales are common mycorrhizal associates of 55:595–606
Ericaceae. New Phytol 174:864–878 Stanghellini ME, Rasmussen SL (1994) Hydroponics—a solution for
Serfling A, Wirsel SGR, Lind V, Deising HB (2007) Performance of zoosporic pathogens. Plant Dis 78:1129–1138
the biocontrol fungus Piriformospora indica on wheat under Stein E, Molitor A, Kogel KH, Waller F (2008) Systemic resistance in
greenhouse and field conditions. Phytopathology 97:523–531 Arabidopsis conferred by the mycorrhizal fungus Piriformospora
Shahollari B, Varma A, Oelmuller R (2005) Expression of a indica requires jasmonic acid signaling and the cytoplasmic
receptor kinase in Arabidopsis roots is stimulated by the basidio- function of NPR1. Plant Cell Physiol 49:1747–1751
mycete Piriformospora indica and the protein accumulates in Talboys PW (1984) Chemical control of verticillium wilts. Phytopathol
Triton X-100 insoluble plasma membrane microdomains. J Plant Mediterr 23:163–175
Physiol 162:945–958 Ton J, Van Pelt JA, van Loon LC, Pieterse CMJ (2002) Differential
Shahollari B, Vadassery J, Varma A, Oelmuller R (2007) A leucine-rich effectiveness of salicylate-dependent and jasmonate/ethylene-
repeat protein is required for growth promotion and enhanced seed dependent induced resistance in Arabidopsis. Mol Plant Microbe
production mediated by the endophytic fungus Piriformospora Interact 15:27–34
indica in Arabidopsis thaliana. Plant J 50:1–13 Vadassery J, Ritter C, Venus Y, Camehl I, Varma A, Shahollari B,
Sharma M, Schmid M, Rothballer M, Hause G, Zuccaro A, Imani J, Novak O, Strnad M, Ludwig-Muller J, Oelmuller R (2008) The
Kampfer P, Domann E, Schäfer P, Hartmann A, Kogel KH role of auxins and cytokinins in the mutualistic interaction
(2008) Detection and identification of bacteria intimately between Arabidopsis and Piriformospora indica. Mol Plant
associated with fungi of the order Sebacinales. Cell Microbiol Microbe Interact 21:1371–1383
10:2235–2246 Van der Vlugt RAA, Cuperus C, Vimk J, Stijger CCMM, Naaldwijk
Shaul O, Galili S, Volpin H, Ginzberg I, Elad Y, Chet I, Kapulnik Y AA, Lesemann DE, Verhoeven JTJ, Roenhorst JW (2002)
(1999) Mycorrhiza-induced changes in disease severity and PR Identification and characterization of Pepino mosaic potexvirus
protein expression in tobacco leaves. Mol Plant Microbe Interact in tomato. EPPO Bull 32:503–508
12:1000–1007 van Loon LC (1997) Induced resistance in plants and the role of
Sherameti I, Shahollari B, Venus Y, Altschmied L, Varma A, pathogenesis-related proteins. Eur J Plant Pathol 103:753–765
Oelmuller R (2005) The endophytic fungus Piriformospora Varma A, Verma S, Sudah SN, Franken P (1999) Piriformospora
indica stimulates the expression of nitrate reductase and the indica, a cultivable plant growth-promoting root endophyte. Appl
starch-degrading enzyme glucan-water dikinase in tobacco and Environ Microbiol 65:2741–2744
Arabidopsis roots through a homeodomain transcription factor Verma S, Varma A, Rexer K-H, Hassel A, Kost G, Sarbhoy A, Bisen
that binds to a conserved motif in their promoters. J Biol Chem P, Bütehorn B, Franken P (1998) Piriformospora indica, gen.
280:26241–26247 nov. sp. nov., a new root-colonizing fungus. Mycologia 90:896–
Sherameti I, Venus Y, Drzewiecki C, Tripathi S, Dan VM, Nitz I, 903
Varma A, Grundler FM, Oelmuller R (2008) PYK10, a beta- Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, Fischer M,
glucosidase located in the endoplasmatic reticulum, is crucial for Heier T, Huckelhoven R, Neumann C, von Wettstein D,
the beneficial interaction between Arabidopsis thaliana and the Franken P, Kogel KH (2005) The endophytic fungus Piriformo-
endophytic fungus Piriformospora indica. Plant J 54:428–439 spora indica reprograms barley to salt-stress tolerance, disease
Shittu HO, Castroverde DCM, Nazar RN, Robb J (2009) Plant- resistance, and higher yield. Proc Natl Acad Sci USA 102:13386–
endophyte interplay protects tomato against a virulent Verticil- 13391
lium. Planta 229:415–426 Waller F, Mukherjee K, Deshmukh SD, Achatz B, Sharma M, Schäfer
Sinha AK, Wood RKS (1968) Studies on nature of resistance in tomato P, Kogel KH (2008) Systemic and local modulation of plant
plants to Verticillium albo-artrum. Ann Appl Biol 62:319–327 responses by Piriformospora indica and related Sebacinales
Sirrenberg A, Goebel C, Grond S, Czempinski N, Ratzinger A, species. J Plant Physiol 165:60–70
Karlovsky P, Santos P, Feussner I, Pawlowski K (2007) Weiss M, Selosse MA, Rexer KH, Urban A, Oberwinkler F (2004)
Piriformospora indica affects plant growth by auxin production. Sebacinales: a hitherto overlooked cosm of heterobasidiomycetes
Physiol Plant 131:581–589 with a broad mycorrhizal potential. Mycol Res 108:1003–1010