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ORIGINAL ARTICLE
Involvement of metabolic components, volatile compounds, PR
proteins, and mechanical strengthening in multilayer
protection of cucumber plants against Rhizoctonia solani activated
by Trichoderma atroviride TRS25
Justyna Nawrocka
1
&U. Małolepsza
1
&K. Szymczak
2
&M. Szczech
3
Received: 25 May 2017 /Accepted: 16 August 2017 /Published online: 6 September 2017
#The Author(s) 2017. This article is an open access publication
Abstract In the present study, the spread of Rhizoctonia
solani-induced disease was limited when cucumber plants
were pretreated with Trichoderma atroviride TRS25. The sys-
temic disease suppression was related to TRS25-induced re-
sistance (TISR) induction with simultaneous plant growth
promotion. Protection of cucumber was related to enhanced
activity of defense enzymes, e.g., guaiacol peroxidase (GPX),
syringaldazine peroxidase (SPX), phenylalanine ammonia ly-
ase (PAL), and polyphenol oxidase (PPO) as well as phenolic
(PC) concentration increases in the conditions of hydrogen
peroxide (H
2
O
2
) accumulation, resulting in thiobarbituric acid
reactive substance (TBARS) decrease. Moreover, the obtained
results indicated that TISR might depend on accumulation of
salicylic acid derivatives, that is methyl salicylate (MeSA),
ethylhexyl salicylate (EHS), salicylic acid glucosylated con-
jugates (SAGC), and β-cyclocitral as well as volatile organic
compounds (VOC) such as Z-3-hexanal, Z-3-hexenol, and
E-2-hexenal. The results point to important, not previously
documented, roles of these VOC in TISR signaling with up-
regulation of PR1 and PR5 gene characteristic of systemic
acquired resistance (SAR) and of PR4 gene, marker of in-
duced systemic resistance (ISR). The study established that
TRS25 enhanced deposition of callose and lignin in special-
ized plant cells, which protected vascular system in cucumber
shoots and roots as well as assimilation cells and dermal tis-
sues in shootsand leaves. These compounds protected cucum-
ber organs against R. solani influence and made them more
flexible and resilient, which contributed to better nutrition and
hydration of plants. The growth promotion coupled with sys-
temic mobilization of biochemical and mechanical strength-
ening might be involved in multilayer protection of cucumber
against R. solani activated by TRS25.
Keywords Callose .Genes .Lignin .Rhizoctonia solani .
Trichode rma .Volatile compounds
Introduction
Rhizoctonia solani is one of the most destructive soil-borne
necrotrophs. The pathogen induces damping-off, blight, rot of
roots, and shoots in a variety of crop plants including cucum-
ber (Singh et al. 2002; Bartz et al. 2010; Saberi et al. 2013;
Yousef et al. 2013). Management of the disease caused by
R. solani is difficult because of great variability in the patho-
gen population and long-term survival in soil (Bartz et al.
2010; Taheri and Tarighi 2012). Chemical fungicides, mainly
methyl bromide together with different cultural practices such
as crop rotation, and methods that minimize contact of the
plant with R. solani are not sufficiently effective (Melo and
Faull 2000; Montealegre et al. 2003). Integrated protection
against the pathogen includes biological control as an impor-
tant alternative or component of the disease management
(Montealegre et al. 2003; Yousef et al. 2013).
Fungal species of the genus Trichoderma, being preva-
lent in soil, have been extensively used as biological con-
trol agents (BCA) against a wide range of plant pathogens
Handling Editor: Bhumi Nath Tripathi
*Justyna Nawrocka
jnawrocka@biol.uni.lodz.pl
1
Department of Plant Physiology and Biochemistry, Faculty of
Biology and Environmental Protection, University of Lodz, Banacha
12/16, 90-237 Lodz, Poland
2
Instituteof General Food Chemistry, Lodz University of Technology,
Stefanowskiego 4/10, 90-237 Lodz, Poland
3
Research Institute of Horticulture, Konstytucji 3 Maja 1/3,
96-100 Skierniewice, Poland
Protoplasma (2018) 255:359–373
DOI 10.1007/s00709-017-1157-1
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
(Harman et al. 2004;Alfanoetal.2007; Hermosa et al.
2012; Zhang et al. 2016). Trichoderma employ several
modes of action contributing to their biocontrol activity
and these modes vary depending on the strain and environ-
ment. Some strains act directly against pathogens via
mycoparasitism, competition, and antibiosis (Elad 2000;
Melo and Faull 2000;Vinaleetal.2008;Monfiland
Casas-Flores 2014; Vos et al. 2015); others promote plant
growth (Harman et al. 2004; Aly and Manal 2009;
Christopher et al. 2010; Yadav et al. 2011)orinducenatu-
ral plant protection at the site of infection as well as at
distance to the pathogen resulting in alteration of the plant
systemic resistance (Harman et al. 2004; Singh et al. 2010;
Hermosa et al. 2012;Yousefetal.2013; López-Bucio et al.
2015; Vos et al. 2015;Zhangetal.2016). Simultaneous
cucumber growth promotion and up-regulation of systemic
resistance against pathogens are observed less frequently
and still need elucidation (Shoresh et al. 2005;Harman
et al. 2012;Mathysetal.2012).
In the previous studies, Trichoderma strains were listed
among the effective mycoparasitic and antagonistic BCA of
R. solani (Khara and Hadwan 1990; Harman et al. 2004;
Yousef et al. 2013). Further analyses suggested that
Trichode rma-induced resistance (TISR), rather than a direct
antifungal influence against R. solani protected plants against
this pathogen (Yousef et al. 2013). Since the necessity to
investigate multifunctional Trichoderma strains simulta-
neously promoting plant growth, inhibiting pathogens, and
enhancing plant protective barriers is emphasized (Shoresh
et al. 2005;Harmanetal.2012; López-Bucio et al. 2015),
the primary aim of the present study was to investigate the
basis of the effective protection of cucumber plants against
R. solani induced by newly identified Trichoderma atroviride
TRS25 strain. Biochemical, molecular, and structural re-
sponses were studied as symptoms of TRS25-induced resis-
tance (TISR). Starting from the biochemical protection, gen-
eration of hydrogen peroxide (H
2
O
2
) which may act as an
antimicrobial agent, signaling molecule, and substrate for de-
fense enzymes (Małolepsza and Różalska 2005; Nanda et al.
2010) was analyzed. Simultaneous studies focused on deter-
mination of the activities of antioxidant enzymes, including
ascorbate (APX), guaiacol (GPX), and syringaldazine perox-
idase (SPX) which maintain the content of reactive oxygen
species (ROS) at a safe level and participate in mechanical
strengthening of plant tissues (Nikraftar et al. 2013).
Moreover, the studies aimed with phenylalanine ammonia
lyase (PAL) and polyphenol oxidase (PPO) involved in the
synthesis of antioxidant and antimicrobial active phenolic
compounds (PC). Among PC, ortodihydroxyphenolics
(oDP), phenylpropanoids (PP), favonoids (FL), and salicylic
acid (SA) were studied as compounds which may contribute
to resistance induction, direct suppression of pathogens, and
to creation of structural barriers which prevent progress of the
disease (Yedida et al. 2003; Park et al. 2007; Singh et al.
2011; Oliveira et al. 2016). To assess membrane stability,
thiobarbituric acid reactive substance (TBARS) concentration
was analyzed. Particular attention was paid to volatile com-
pounds (VOCs) including methyl salicylate (MeSA),
ethylhexyl salicylate (EHS), and β-cyclocitral as well as un-
saturated fatty acids, that is, linolenic and linoleic acid deriv-
atives, including Z-3-hexanal, Z-3-hexenol, and E-2-hexenal
not studied extensively as compounds which together with
H
2
O
2
may be involved in resistance signaling resulting in
enhanced expression of defense genes of pathogenesis-
related (PR) proteins (Mathys et al. 2012; Brotman et al.
2013;Dudarevaetal.2013; Gao et al. 2014;Lvetal.2015;
De Palma et al. 2016; Adrian et al. 2017). Except of MeSA,
these compounds were not considered among metabolic com-
ponents playing the role of signaling molecules in plants in
response to Trichoderm a influence. To characterize the TISR
induced by the detected VOC, studies of gene transcript ex-
pression were performed for PR1 (acidic proteins) and PR5
(thaumatin-like proteins) marker genes of the systemic ac-
quired resistance (SAR) and for PR4 (chitinase) and PR12
(defensin) gene characteristic of induced systemic resistance
(ISR). The tested genes are known as allowing to determine
kind of resistance, basically, JA- and SA-dependent or both
(Alfano et al. 2007; Salas-Marina et al. 2011; Hermosa et al.
2012; Perazzolli et al. 2012; Martinez-Medina et al. 2013;
Nawrocka and Małolepsza 2013; Vos et al. 2015), which
might be induced by the tested Trichoderma. Finally, we
particularly focused on the deposition and location of stabi-
lizing components such as callose and phenolic polymer, lig-
nin important for plant mechanical strengthening. The struc-
tural barriers in plant cells may be related to crosslinking of
proteins and deposition of the mentioned compounds to sep-
arate and protect susceptible tissues, to limit spread of the
pathogen, or to delay the infection process until other defen-
sive mechanisms become active (Solanki et al. 2011;
Nikraftar et al. 2013; Rao et al. 2015). A common model of
callose synthesis favors a deposition of this glucan in the
paramural space between a cell wall and a plasma membrane
in the form of papillae while lignin usually builds secondary
walls of root vessel and dermal tissue cells in the area of
pathogen influence (Chowdhury et al. 2014; Rao et al.
2015; Schneider et al. 2016). Since enhancement of structural
barriers was mainly detected and analyzed in the tissues di-
rectly attacked by pathogens, we focused on systemic loca-
tion of callose and lignin in the entire plant and their role not
only in the protection against R. solani but also in cucumber
growth promotion. Identification of the basis of TRS25-
induced cucumber resistance including metabolic compo-
nents, VOC, PR proteins, and mechanical strengthening with
simultaneous plant growth promotion would aid to elucidate
the complicated network activated by this strain effectively
protecting cucumber plants against R. solani.
360 J. Nawrocka et al.
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Material and methods
Fungal strains and plant material preparation
T. atro vi ri de TRS25 was collected from a growing medium
for mushroom production. TRS25 was confirmed as not path-
ogenic for the cultivated mushroom. The isolate was identified
morphologically using an interactive key (http://nt.ars-grin.
gov/taxadescriptions/keys/TrichodermaIndex.cfm),
supported by identification keys provided by Gams and
Bissett (1998)andSamuelsetal.(2002). The identification
was followed by molecular classification of the isolate per-
formed by Oskiera et al. (2015). The microorganism se-
quences have been deposited at the NCBI GenBank (http://
www.ncbi.nlm.nih.gov) with accession numbers: ITS
KJ786731 and tef1αKJ786812.
Complementary tests examining antagonistic or
mycoparasitic properties of TRS25 isolate against various
pathogens including R. solani showed moderate ability of this
strain to colonize, overgrow, or parasitize fungal sclerotia
(Szczech et al. 2014). TRS25 isolate used to treat cucumber
plants was grown on Malt Extract Agar media (Fluka) in petri
plates for 10 days at 25 °C. Every 24 h, the cultures were
exposed to light for 20 min to activate fungus sporulation.
To obtain inoculum of TRS25, the spores of the fungus were
washed off the surface of 10-day old cultures with 10 ml of
0.85% NaCl solution. R. solani Kühn MUCL47938 strain
used in the experiment is a well-characterized, standard phy-
topathogen of cucumber plants. The fungus was grown for
7 days in 9-cm diameter petri plates on potato dextrose agar
(PDA), in the dark, at the constant temperature of 25 °C. After
incubation, mycelia mats of five plates were homogenized in
0.5 l of deionized water and used to inoculate cucumber
plants.
Cucumber (Cucumis sativus L.) plants cv. Iwa F
1
,suscep-
tible to R. solani, were used in the experiment. The plants were
cultivated in the media consisting of podsolic soil and vermic-
ulite 1:1 (v:v). To investigate the potential of TRS25 to induce
resistance in cucumber plants, the growing medium was sup-
plemented with aliquots of the spore suspension to obtain 10
6
spore density per 1 g of the medium. The medium was thor-
oughly mixed and distributed to 0.5-dm
3
plastic pots. Medium
without TRS25 was used as control. Approximately 500 g of
the medium with 60% water holding capacity adjusted with
tap water was used in each pot and sown with one seed of
cucumber per pot. Cucumber plants grew in a chamber
(Sanyo, model MLR-351 H with 15 fluorescent lamps type
FL 40SSW/37), under 14-h photoperiod with a 20,000-lx light
intensity at 25/20 °C, day/night temperature cycle, and 80%
relative humidity. The plants were watered daily with tap wa-
ter. Seven-day-old cucumber seedlings were fertilized with
Polyfeed (POLY-FEED VITA 12-10-34+2 HAIFA, Poland).
Three weeks after sowing half of the control and TRS25,
pretreated plants were inoculated with 5 ml of the R. solani
mycelia homogenate near the cucumber stem base according
to the method of this pathogen application presented by
Pannecoucque and Höfte (2009). All plants were then main-
tained in the growth chamber for 7 days. Four experimental
groups of plants were tested: (I) control plants—nontreated
with TRS25 and uninoculated with R. solani,(II)Rs
plants—nontreated with TRS25 and inoculated with
R. solani,(III)TRS25plants—pretreated with TRS25, unin-
oculated with R. solani, and (IV) TRS25 + Rs plants—TRS25
pretreated, challenged with R. solani. Twelve pots were pre-
pared for each treatment. The experiment was prepared four
times under the same conditions. Twenty-eight-day old cu-
cumber plants were cut at the stem base. Their fresh weight
(FW) was determined, and the third leaf of each plant was cut
off for biochemical assays. In each experiment, the samples
for biochemical analyses were prepared in triplicate. The ir-
regular lesions and rot symptoms were observed on the roots
of 5-week old Rs plants. Disease development on the root
surfaces was evaluated using a Motic Images Plus 2.0
ML
pro-
gram (Motic China Group, Asia) according to the manufac-
turer’s instruction. Then, the roots were dried at 60 °C for 24 h
and their dry weight (DW) was measured.
Hydrogen peroxide (H
2
O
2
), thiobarbituric acid reactive
substance, and protein contents
To measure H
2
O
2
content, the compound was extracted from
500-mg samples from three fresh leaves according to the mod-
ified method of Capaldi and Taylor (1983), previously de-
scribedbyMałolepsza and Różalska (2005). The content
was calculated based on the standard curve of H
2
O
2
and
expressed in μmol per g of FW.
As an index of lipid peroxidation, the TBARS content was
determined according to the method ofYagi (1976)withmod-
ifications, as described by Nawrocka et al. (2011). TBARS
content was estimated by referring to a standard 1,1,3,3-
tetraethoxypropane (Sigma-Aldrich) and expressed in nmol
per g of FW. The supernatants prepared for TBARS determi-
nation were also used to study protein content according to the
method of Bradford (1976) with a standard curve prepared
using bovine serum albumin (Sigma-Aldrich).
Enzymatic activity
To measure ascorbate, guaiacol, and syringaldazine peroxi-
dase (APX, EC.1.11.1.11; GPX, EC 1.11.1.7 and SPX, EC
1.11.1.7, respectively) activities, 250-mg samples from three
fresh leaves were ground in an ice-cold mortar with 50-mM
sodium phosphate buffer (1:5; w:v; pH 7.0) containing 1-mM
EDTA, 1-mM sodium ascorbate, and 0.5-M NaCl and centri-
fuged (15,000 ×g, 15 min). APX activity was assayed using
the modified method of Nakano and Asada (1981)following
Involvement of metabolic components, volatile compounds, PR proteins, and mechanical strengthening in... 361
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
the oxidation of ascorbate to dehydroascorbate, which was
determined at 265 nm. The enzyme activity was calculated
based on absorbance coefficient ε= 13.7 mM
−1
cm
−1
and
expressed in units (1 U = 1 μmol ascorbate oxidized
min
−1
mg
−1
protein). GPX activity was estimated by measur-
ing an increase in absorbance of tetraguaiacol (ε=26.6
mM
−1
cm
−1
), a colored product of guaiacol oxidation, at
470 nm for 4 min at 30 °C (Maehly and Chance 1954;
Nawrocka et al. 2011). The enzyme activity was expressed
in units (1 U = 1 mmol of tetraguaiacol formed min
−1
mg
−1
protein). SPX activity was assayed by the method of Imberty
et al. (1985) following oxidation of syringaldazine
(ε=27mM
−1
cm
−1
). The absorbance increase was measured
at 530 nm and the enzyme activity was expressed in units
(1 U = 1 μmol of syringaldazine oxidized min
−1
mg
−1
protein).
To determine phenylalanine ammonia lyase (PAL; EC
4.3.1.24) activity, 500-mg samples from three fresh leaves
were ground in an ice-cold mortar with 0.5-M Tris-HCl buffer
(1:10; w:v; pH 8.8), containing 0.8-mM β-mercaptoethanol
and 1% polyvinylpolypyrrolidone. PAL was determined ac-
cording to Zucker (1965) by the production of trans-cinnamic
acid from l-phenylalanine during 1 h at 37 °C. The enzyme
activity was expressed in units (1 U = 1 μmol of trans-
cinnamic acid formed min
−1
mg
−1
protein). To measure poly-
phenol oxidase (PPO; EC 1.10.3.1) activity, 500-mg samples
from three fresh leaves were ground in an ice-cold mortar with
50-mM sodium phosphate buffer (1:5; w:v; pH 7.0) containing
1-M NaCl, stirred for 30 min at room temperature and centri-
fuged (20,000 ×g, 20 min). PPO activity was determined ac-
cording to the method of Chang et al. (2000) by measuring the
increase in absorbance at 420 nm following the oxidation of
catechol. One unit of PPO activity was calculated based on
absorbance coefficient ε=2.72mM
−1
cm
−1
and expressed in
units (1 U = 1 nmol catechol oxidized min
−1
mg
−1
protein).
Phenolic compound content
To determine PC, oDP, PP, and flavonoid (FL) contents 500-
mg samples from three frozen leaves were homogenized with
80% MeOH (1:10; w:v) and centrifuged (20,000 ×g,20min).
PC content was determined by the method of Singleton and
Rossi (1965) and oDP were determined with Johnson and
Shaal (1957) method, as described previously by Nawrocka
et al. (2011). PC and oDP contents were calculated using a
standard curve prepared for chlorogenic acid (Sigma-Aldrich)
and expressed in mg per g FW. PP content was measured in
the crude extract obtained by mixing the supernatant with
0.1% HCl in 95% ethanol and 2% HCl, 1:18 (v:v)according
to Glories (1978). The absorbance at 320 nm was measured.
PP content was calculated on the basis of a standard curve of
the caffeic acid (Sigma-Aldrich) and expressed in mg per g of
FW. FL content was estimated using the modified method of
Chang et al. (2002). The reaction mixture containing metha-
nolic extract, 10% AlCl
3
×6H
2
O, and 1-M CH
3
COONa was
incubated for 30 min at room temperature. The absorbance
was measured at 234 nm. FL content was expressed in mg
of the standard quercetin (5 mg/100 ml) (Sigma-Aldrich) per g
of FW.
Salicylic acid and salicylic acid glucosylated conjugate
contents
SAwas extracted according to the modified protocol of Molina
et al. (2002). One-gram samples from frozen leaves were ex-
tracted three times in 80, 90, and then 100% (1:10; v/v) MeOH.
After centrifugation (15 min, 20,000 ×g), all the supernatants
were combined and evaporated to dryness under vacuum at
65 °C. The residue was redissolved in water at 80 °C and
centrifuged (15 min, 20,000 ×g). Then SAwas extracted three
times into three volumes of cyclopentane:ethylacetate:2-
propanol (50:50:1, v/v/v). The organic extracts were dried un-
der vacuum and resuspended in 70% MeOH containing 0.5%
fumaric acid (FA). To release SA from SAGC, the aqueous
phase was acidified with HCl to pH 1.5–2.0 and boiled for
1.5 h at 80 °C. The released SAwas extracted with the organic
mixture as described above. HPLC system (DIONEX,
Sunnyvale, CA, USA) was used for analysis. Separation of
SA took place over an RP column (aQ Hypersil GOLD,
250 mm × 4.6 mm, 5 μm) joined with a guard column
(GOLD aQ Drop-In guards, 10 mm × 4 mm, 5 μm) at 40 °C,
by use of a binary solvent system consisting of (A) water and
(B) methanol with 0.5% FAwith a flow rate of 1.5 ml/min. The
elution profile was as follows: 0–2 min, 40%B; 2–10 min, 40–
60% B; 10–12 min, 60% B; 12–13 min, 60–40% B; and 13–
15 min, 40% B. Chromatograms were obtained by fluores-
cence evaluation (excitation 301 nm, emission 412 nm).
Quantification was based on the calibration curves for the ad-
equate SA standards (Sigma-Aldrich) according to the modi-
fied method of Seskar et al. (1998).
Volatile compound content
Solid-phase microextraction (SPME) used for determination
of VOC was carried out with the method of Carlin et al.
(2016). Three grams of freshly harvested leaves was incubated
in 20-ml headspace vials at 40 °C for 30 min. Subsequently,
extraction was performed using 50/30 divinylbenzene/
carboxen/polydimethylsilox (DVB/CAR/PDMS) 1-cm long
fiber for 60 min and after that the samples were introduced
into gas chromatograph injection port and desorbed at 240 °C
with a split ratio 1:30. All samples were analyzed by GCxGC
TOF-MS. Analysis was performed using Pegasus 4D mass
spectrometer equipped with a consumable-free dual-stage,
quad-jet thermal modulator (LECO Corp.). A BPX5 (30 m,
0.25 mm, 0.25 μm)wasusedasafirst-dimension(1D)
362 J. Nawrocka et al.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
column, and a BPX50 (2 m, 0.1 mm, 0.1 μm) was used as a
second-dimension (2D) column. The carrier gas was helium,
constant flow 1 ml/min. Temperature program conditions
were as follows: first oven +50 (3 min)–280 °C at 4 °C/min,
second oven, and modulator, respectively, +5 and +20 °C rel-
atively to the first oven program (modulation time 8 s, hot
pulse time 2.4 s, cold pulse −80 °C, time 1.6 s). TOF mass
spectrometer parameters included mass range of m/z 33–
550 at 30 spectra/s, ionization energy 70 eV, and ion source
temperature 200 °C. The relative VOC content was estimated
on the basis of peak areas obtained for these compounds in the
control, Rs, TRS25, and TRS25 + Rs plants.
qRT-PCR analysis of defense genes
of pathogenesis-related proteins
To evaluate expression of defense-related genes of PR pro-
teins, 200 mg of fresh cucumber leaves was frozen in liquid
nitrogen and grind immediately. RNA was extracted individ-
ually from the samples of six replicates for each treatment.
Total RNA was extracted with RNAzol® RT (Sigma-
Aldrich) according to the instruction of the manufacturer with
modifications. To precipitate RNA, supernatant was mixed
with one volume of the mixture of isopropanol and NaCl-
citrate buffer (0.2-M NaCl, 0.02-M sodium citrate) (1:1;
w:w) to remove sugars, phenolics, and pollutants. The
denaturing electrophoresis according to Masek et al. (2005)
was performed to confirm the integrity of RNA structure.
Extracted RNA was treated with DNase (Ambion).
Subsequently, DNA-free total RNA was converted into
cDNA using TranScriba Kit (A&A Biotechnology) with
oligo-dT primers according to the manufacturer’s instructions.
The primers used in qRT-PCR analysis are presented in
Table 1. PCR reactions were carried out in 200-μleppendorfs.
The reaction mixture contained 3 μl of diluted cDNA, 9 μlof
real-time 2xPCR Master Mix SYBR® A (A&A
Biotechnology), 4-μl primers mix (oligo.pl), and 4-μlDEPC
water. Efficiency of primers was calculated by the dilution
method. Quantitative real-time PCR was performed using
the Rotor-Gene® Q Detection System at 95 °C for 5 min
followed by 40 cycles at 95 °C for 15 s and at 60 °C for
1 min. UBI-ep (Ubiquitin extension protein) and TUA (α-
Tubulin) genes were chosen as the most stable reference genes
in the used analytical system. Relative expression of genes of
interest was calculated according to Pfaffl (2001).
Callose and lignin deposition and location
To determine callose content, 500 mg of cucumber fresh
leaves was homogenized with 95% EtOH (1:5; w:v) and cen-
trifuged (15,000 ×g, 20 min). The supernatant was discarded
and the pellet was dissolved in 0.4 ml of 1-M NaOH and
incubated at 80 °C for 30 min to solubilize the callose. After
centrifugation at room temperature (12,000 ×g, 20 min), the
supernatant was used for callose determination which was
performed according to Kőhle et al. (1985). The reaction mix-
ture contained 0.4 ml of the supernatant, 0.8-ml 0.1% aniline
blue, 0.42-ml 1-M HCl, and 1.18-ml 1.0-M glycine-NaOH.
After incubation at 50 °C for 30 min and subsequently at room
temperature for 30 min under shaking conditions, fluores-
cence was quantified at an excitation wavelength of 400 nm
and an emission wavelength of 485 nm. Amounts of callose
were expressed as Euglena gracilis β-1,3-glucan (Sigma-
Aldrich) equivalent in mg per g of FW. For histochemical
detection of callose deposition, plant tissue fragments were
washed with 25-mM phosphate buffer (pH 8.2) and stained
with 0.1% aniline blue prepared with 100-mM phosphate
buffered saline (PBS) (pH 8.2) for approximately 20 min ac-
cording to Kaźmierczak (2008). The tissue fragments were
then immediately viewed in a fluorescent confocal micro-
scope using a UV filter and excitation and emission wave-
lengths of 480 and 570 nm, respectively.
Lignin compounds were extracted according to the method
given by Bruce and West (1989) and Mandal (2010) with
modifications. Five hundred milligrams of cucumber tissue
was homogenized in 80% MeOH (1:10; w:v) and centrifuged
(15,000 ×g, 20 min). The pellet was washed three times with
80% MeOH and dried at 60 °C for 24 h. Fifteen milligrams of
the insoluble residue was mixed with 5 ml of 2-M HCl and
0.5 ml of TGA, boiled in water bath for 4 h, and centrifuged
Table 1 Gene-specific primer pairs used in the qRT-PCR experiment
Gene Forward primer Reverse primer Reference
PR 4 5′-TGGTCACTGCAACCCTGACA-3′5′-AGTGGCCTGGAATCCGACT-3′Alizadeh et al. 2013
PR 12 5′-AATGGATCCATGGCTAAGTTGCTTCCATC-3′5′-AATGAATTCAATACACACGATTTAGCACC-3′Hwangbo et al. 2016
PR 1 5′-TGCTCAACAATATGCGAACC-3′5′-TCATCCACCCACAACTGAAC-3′Alizadeh et al. 2013
PR 5 5′-CATTCTGCCTTTGTGCTTTTTC-3′5′-ATTGATCGTCACGGTCTCGCC-3′Liu et al. 2015
Reference gene
UBI-ep 5′-CACCAAGCCCAAGAAGATC-3′5′-TAAACCTAATCACCACCAGC-3′Wan et al . 2010
TUA 5′-ACGCTGTTGGTGGTGGTAC-3′5′-GAGAGGGGTAAACAGTGAATC-3′Wan et a l. 2010
Involvement of metabolic components, volatile compounds, PR proteins, and mechanical strengthening in... 363
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
(20 min, 10,000 ×g). The supernatant was drained out. The
pellet was washed two times in distilled water, then suspended
in 2.5 ml of 0.5-M NaOH, and allowed to shake on an orbital
shaker for 2 h at 25 °C followed by centrifugation (20 min,
10,000 ×g). The supernatant was then mixed with 1 ml of
concentrated HCl and thioglycolic acid (TGA) (90:1; v:v)
and allowed to precipitate at 4 °C for 4 h. An orange brown
pellet obtained after discarding the supernatant was dissolved
in 0.5-N NaOH. After absorbance measuring at 280 nm, the
quantification was based on the calibration curve for lignin
standard (alkali, 2-hydroxy-propyl ether) (Sigma-Aldrich)
and the content was expressed as μgpergofFW.
Histochemical detection of lignin deposition was prepared
according to Drnovšek et al. (2005). Plant tissue fragments
were washed with 80% EtOH and stained with 1% acridine
orange prepared with 96% EtOH for approximately 30 min.
Then, the samples were washed in 25% HCl.The tissue frag-
ments were then immediately viewed in a fluorescent confocal
microscope using a UV filter and excitation and emission
wavelengths of 488 and 540 nm, respectively.
Statistical analyses
To determine cucumber shoot FWand root DW as well as to
analyze biochemical parameters, the results from four inde-
pendent, not significantly different trials, were combined. In
all experiments, six replicates for each variant were obtained.
Statistical analysis of variance (ANOVA, P< 0.05) for each
parameter was followed by the Duncan multiple range post
hoc test. Respective significant differences were marked using
letters a, b,c, and d.The analyses were performed inStatistica,
Version 10: New Features and Enhancements.
Results
Impact of Trichoderma on R. solani infection
and cucumber growth
R. solani effectively infected cucumber plants nontreated with
Trichoderma (Rs plants). Irregular brown lesions and rot
symptoms on the roots of these plants were followed by shoot
and leaf dark brown blight blotches and plant collapse (Fig. 1).
The development of disease symptoms in the roots of Rs
plants oscillated approximately 75% above the uninfected
control (Fig. 2a). Rs plants had underdeveloped and dense
root system, which established average 40% lower DW in
comparison to the control plants (Fig. 2c). The significant
disease suppression down to mean 18% of the root area was
observed in the plants pretreated with T. at roviri de TRS25 and
inoculated with R. solani (TRS25 + Rs plants) as compared to
Rs plants (Fig. 2a). Simultaneously, TRS25 suppressed nega-
tive effect of R. solani on cucumber growth and plant collapse
(Fig. 1). Both TRS25 and TRS25 + Rs plants had better de-
veloped root system and above ground parts. TRS25 caused
pronounced increase in FW of the shoots on average by 55 and
82% (Fig. 2b) and increase in DW of the roots on average by
77 and 96% (Fig. 2c) in TRS25 and TRS25 + Rs plants as
compared to the control and Rs plants, respectively.
Biochemical, molecular, and structural hallmarks of TISR
against R. solani induced in cucumber by TRS25
R. solani inoculation significantly enhanced TBARS concen-
tration (Table 2a) and decreased APX activity in Rs plants as
compared to the control (Table 2b). Treatment of cucumber
with TRS25 increased H
2
O
2
content and decreased that of
TBARS in TRS25 and TRS25 + Rs plants as compared to
the control and Rs plants (Table 2a). Simultaneously, in the
same plants, TRS25 significantly decreased APX activity and
increased PAL and PPO activities, while no significant differ-
ences were detected for these parameters between TRS25 and
TRS25 + Rs variants. Moreover, in TRS25 + Rs plants, addi-
tional enhancement of GPX and SPX activities was observed
as compared to the control, Rs, and TRS25 plants (Table 2b).
Along with induction of PAL and PPO activities, accumula-
tion of PC in the form of oDP and PP both in TRS25 and
Fig. 1 Suppression of R. solani-induced disease spreading on cucumber
roots, shoots, and leaves by TRS25. Irregular brown lesions and rot
symptoms on the roots of Rs plants were followed by shoot and leaf
dark brown blight blotches and plant collapse. Abbreviations: Rs plants,
TRS25 nontreated, inoculated with R. solani; TRS25 + Rs plants, TRS25
pretreated, inoculated with R. solani
364 J. Nawrocka et al.
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TRS25 + Rs plants and accompanied with the increase in FL
in the TRS25 + Rs group as compared to the control and Rs
plants was detected (Table 2c). The strongest, above twofold,
increase was observed for PP content in TRS25 + Rs plants as
compared to Rs plants. In the PP group, SAGC concentration
increased both in TRS25 and TRS25 + Rs plants as compared
to the control and Rs plants (Table 2c). R. solani inoculation
induced generation of Z-3-hexanal and E-2-hexenal, two
VOC compounds which were not detected in the control
plants and it increased Z-3-hexenol concentration as com-
pared to the control (Table. 2c). The significant increases in
Z-3-hexanal and E-2-hexenal accompanied by accumulation
ofMeSA,EHS,andβ-cyclocitral were detected both in
TRS25 and TRS25 + Rs plants, respectively, to the control
and Rs plants (Table 2c). The increase in Z-3-hexenol concen-
tration was additionally observed in TRS25 + Rs plants as
compared to the control (Table 2c). The strongest 2.5-fold
increase in MeSA content was detected in TRS25 plants and
the strongest increases in EHS (3.11-fold), β-cyclocitral
(2.99-fold), Z-3-hexanal (2.14-fold), Z-3-hexenol (1.84-fold),
and E-2-hexenal (2.21-fold) contents in TRS25 + Rs plants.
The molecular analysis revealed that R. solani inoculation
triggered increase in PR5 and PR4 expression in Rs plants
as compared to the control (Fig. 3b and c). Among all tested
genes, TRS25 significantly up-regulated expression of PR1
and PR5 genes in TRS25 and TRS25 + Rs plants as compared
to the control and Rs plants (Fig. 3a and b). The increases of
both genes in TRS25 plants did not differ from those observed
in TRS25 + Rs plants. In the presence of R. solani,additional
PR4 gene expression increase was observed in TRS25 + Rs as
compared to the control and Rs plants (Fig. 3c). In the same
plants, neither TRS25 nor R. solani influenced PR12 gene
expression (Fig. 3d).
Simultaneous to biochemical and molecular response, the
present study demonstrated significant increases inconcentra-
tions of callose and lignin in TRS25 and TRS25 + Rs plants as
compared to the control and Rs plants, while there were no
differences in the accumulation of these compounds between
TRS25 and TRS25 + Rs plants (Table 2d). The changes were
not observed in Rs plants. The microscopic analyses showed
that in TRS25 plants, strong fluorescence signal of callose was
detected in vascular bundles in shoots where the polysaccha-
ride was accumulated as punctate distribution patterns in the
cells separating internal phloem from the shoot pith (Fig. 4a)
as well as in the tissue between xylem vessels of adjacent
vascular bundles in roots (Fig. 4b). Enhanced deposition of
lignin appeared in shoot, root, and leave tissues of
Trichode rma-treated plants. In these plants, the polymer was
accumulated in the outer layer of external phloem in shoot
vascular bundles, while it was not detectable in the control
plants (Fig. 5aandb).Strongaccumulationofligninascom-
pared to the control was also observed in the xylem vessels in
roots as well as in walls of epidermis and collenchyma cells in
shoots (Fig. 5c) and leaves (Fig. 5e).
Discussion
The present study showed that R. solani directly infected cucum-
ber roots. Then, the disease spread systemically in tissues, which
had no contact with the pathogen leading to seedling growth
inhibition and plant collapse. The observed changes were similar
to root rot and foliar blight caused by R. solani,whichwere
described previously in cucumber, rice, and tomato plants
(Singh et al. 2002; Bartz et al. 2010; Yousef et al. 2013;
Małolepsza et al. 2017). Here, we showed that application of
TRS25 significantly reduced disease incidence through activa-
tion of TISR and simultaneously it promoted plant growth. The
ability of the tested TRS25 strain to promote plant growth and to
induce resistance may be explained by the suggestion that some
Fig. 2 Rating of disease caused by R. solani (a) and effect of R. solani
and TRS25 treatments on cucumber shoot fresh weight (FW) (b) and root
dry weight (DW) (c). Values represent the means + SE from three inde-
pendent experiments with six replicates each. Statistical analysis of var-
iance (ANOVA, P< 0.05) for each parameter was followed by the
Duncan multiple range post hoc test. Respective significant differences
were marked using letters a, b, c, and d. Control plants (TRS25
nontreated, uninoculated with R. solani), Rs plants (TRS25 nontreated,
inoculated with R. solani), TRS25 plants (TRS25 pretreated, uninoculat-
ed with R. solani), and TRS25 + Rs plants (TRS25 pretreated, inoculated
with R. solani) were used in the study
Involvement of metabolic components, volatile compounds, PR proteins, and mechanical strengthening in... 365
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Trichoderma strains can activate plant secondary metabolism
simultaneously limiting Bcosts^of primary metabolism. The
chemical substances released into the rhizosphere by
Trichoderma may directly stimulate plant growth and produc-
tivity as well as enhance solubilization and uptake of microele-
ments, nutrients, and water by plants (Harman et al. 2004;
Alfano et al. 2007;Azarmietal.2011; López-Bucio et al.
2015). Moreover, mechanical strengthening of dermal layers
and vascular system induced by TRS25 seem to be crucial for
plant growth promotion, water and nutrient transport improve-
ment and proper turgor maintaining, as well as enhancement of
plant protection against R. solani-induced disease.
The protection of plants by T. atro vi ri de might be triggered
by multiple secondary metabolites released by the fungus,
which influence both pathogens and plants (Contreras-
Cornejo et al. 2009; Tucci et al. 2011; López-Bucio et al.
2015). The preliminary study showed that despite the fact that
TRS25 released different volatiles, it had moderate ability to
directly kill or antagonize R. solani.Itdidnotreducegrowth
and did not overgrow mycelium of this pathogen (Nawrocka
et al. 2011; Szczech et al. 2014).In the present study, TRS25
suppressed the expression of the disease in cucumber roots
where, to a certain extent, it might impede R. solani prolifera-
tion. However, suppression of systemic lesions, blight blotches,
Table 2 Effect of TRS25 on (a) hydrogen peroxide (H
2
O
2
) and thio-
barbituric acid reactive substance, hallmarks of lipid peroxidation
(TBARS) content, (b) ascorbate, guaiacol and syringaldazine peroxidase
(APX, GPX, and SPX), phenylalanine ammonia lyase (PAL) and poly-
phenol oxidase (PPO) activities, (c) phenolics (PC),
ortodihydroxyphenolics (oDP), phenylpropanoids (PP), favonoids (FL),
free salicylic acid (SA), salicylic acid glucosylated conjugates (SAGC),
and volatiles: methyl salicylate (MeSA), ethylhexyl salicylate (EHS) β-
cyclocitral, Z-3-hexanal, Z-3-hexenol and E-2-hexenal content, (d)
callose and lignin content
Biochemical parameter Control Rs TRS25 TRS25 + Rs
a
H
2
O
2
(μmol g
−1
FW) 2.20 ± 0.38 a 2.16 ± 0.31 a 3.25 ± 0.48 b 3.13 ± 0.46 b
TBARS (nmol g
−1
FW) 16.75 ± 4.07 b 20.76 ± 2.44 c 12.43 ± 0.58 a 12.71 ± 3.32 a
b
Defense enzymes
APX(Umg
−1
protein) 2.42 ± 0.55 c 0.90 ± 0.54 a 1.51 ± 0.28 b 1.50 ± 0.31 b
GPX(Umg
−1
protein) 1.00 ± 0.11 a 1.13 ± 0.13 a 0.99 ± 0.07 a 1.52 ± 0.11 b
SPX(Umg
−1
protein) 0.31 ± 0.07 ab 0.39 ± 0.10 b 0.23 ± 0.06 a 0.54 ± 0.04 c
PAL ( U mg
−1
protein) 0.57 ± 0.07 a 0.82 ± 0.11 ab 1.22 ± 0.21 bc 1.46 ± 0.25 c
PPO(Umg
−1
protein) 1.84 ± 0.36 a 2.11 ± 0.17 a 4.04 ± 1.02 c 3.84 ± 0.57 bc
c
Metabolic components
PC (mg g
−1
FW) 4.87 ± 1.01 a 4.37 ± 1.22 a 9.84 ± 2.11 b 9.66 ± 1.15 b
oDP (mg g
−1
FW) 0.76 ± 0.09 a 0.75 ± 0.20 a 0.90 ± 0.05 b 1.11 ± 0.17 b
PP (mg g
−1
FW) 3.22 ± 0.28 a 2.70 ± 0.55 a 5.08 ± 0.76 b 5.40 ± 1.00 b
FL (mg g
−1
FW) 0.25 ± 0.02 a 0.27 ± 0.03 a 0.30 ± 0.03 a 0.37 ± 0.02 b
SA (μgg
−1
FW) 2.98 ± 0.21 a 1.88 ± 0.15 a 2.95 ± 0.34 a 1.98 ± 0.07 a
SAGC (μgg
−1
FW) 2.95 ± 0.31 a 3.12 ± 0.17 a 5.88 ± 0.41 b 6.31 ± 0.19 b
VOC
MeSA (fold) 1.00 ± 0.00 a 1.50 ± 0.01 a 2.50 ± 0.05 c 2.00 ± 0.04 b
EHS (fold) 1.00 ± 0.00 a 1.17 ± 0.13 a 2.25 ± 0.08 b 3.11 ± 0.09 c
β-cyclocitral (fold) 1.00 ± 0.00 a 1.11 ± 0.08 a 3.11 ± 0.12 b 2.99 ± 0.14 b
Z-3-Hexanal (fold) 0.00 ± 0.00 a 1.10 ± 0.00 b 0.98 ± 0.20 b 2.14 ± 0.33 c
Z-3-Hexenol (fold) 1.00 ± 0.00 a 1.50 ± 0.21 bc 1.14 ± 0.17 ab 1.84 ± 0.36 c
E-2-Hexenal (fold) 0.00 ± 0.00 a 1.05 ± 0.00 b 1.10 ± 0.10 b 2.21 ± 0.17 c
d
Structural barriers
Callose (μgg
−1
FW) 300 ± 15 ab 248 ± 20 a 415 ± 17 c 421 ± 7 c
Lignin (mg g
−1
FW) 1.05 ± 0.12 a 1.29 ± 0.05 ab 2.25 ± 0.09 c 1.87 ± 0.08 c
Values represent the means + SE from three independent experiments with six replicates each. Statistical analysis of variance (ANOVA, P< 0.05) for
each parameter was followed by the Duncan multiple range post hoc test. Respective significant differences were marked using letters a, b, c, and d.
Control plants (TRS25 nontreated, uninoculated with R. solani), Rs plants (TRS25 nontreated, inoculated with R. solani), TRS25 plants (TRS25
pretreated, uninoculated with R. solani), and TRS25 + Rs plants (TRS25 pretreated, inoculated with R. solani) were used in the study
366 J. Nawrocka et al.
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and rot symptoms in tissues which were not infected by the
pathogen seemed to be related to TISR as in the case of cucum-
ber, tomato, and Arabidopsis thaliana protected, respectively,
against Pseudomonas syringae,Fusarium oxysporum, and
Botrytis cinerea by different Trichoderma strains (Yedidia
et al. 2003; Ozbay et al. 2004; Mathys et al. 2012).
Enhancement of defense enzyme activities, accumulation of
metabolic components including PC and VOC accompanied
by stimulation of defense gene expression, and structural barrier
strengthening were studied as symptoms of multilayer TISR. It
was showed that TRS25 protected cucumber cells against
membrane destruction and increased H
2
O
2
up to the beneficial
level that allowed it to act as a resistance signaling molecule,
activator of protective genes, and as a substrate for defense
enzymes (Gayoso et al. 2010; Djébali et al. 2011;Harman
et al. 2012; Singh et al. 2011). Simultaneously, as in previous
reports of Singh et al. (2011),Solankietal.(2011), and Surekha
et al. (2013) focusing on different Trichoderma strains, in the
present study lipid peroxidation decrease might result from ad-
ditional protection of cells by GPX and SPX strongly involved
in plant cell wall strengthening during, e.g., lignification as well
as from increase in PC synthesis as a result of enhanced PAL
and PPO enzyme activities induced by TRS25. The important
role of overproduced oDP, PP, and FL in TISR and direct sup-
pression of pathogens as a result of their antioxidative or anti-
biotic properties were presented previously (Shoresh et al.
2005; Lattanzio et al. 2006; Harman et al. 2012; Nikraftar
et al. 2013; Zhang et al. 2016). Moreover, as described by
Gayoso et al. (2010),Solankietal.(2011), and Nikraftar et al.
(2013), PC interacting with PR proteins and defense enzymes
might participate in toughening of cell walls during lignification
process.
In the recent years, it has been documented that TISR in-
duced against different pathogens may involve a complex net-
work of cross-communicating signaling molecules. Most of
the studies focused on the relationship between jasmonic acid
(JA), ethylene (ET), and SA and their derivatives (Yedidia
et al. 2003; Shoresh et al. 2005; Segarra et al. 2007;Salas-
Marina et al. 2011; Tucci et al. 2011; Martinez-Medina et al.
2013;Perazzollietal.2012). The preliminary study showed
no changes in the content of precursors of JA including
octadecanoid and octadecatrienoic acids as well as no signif-
icant synthesis of methyl jasmonate (MeJA) derivative and of
ET in TRS25 and TRS25 + Rs plants (data not shown). On the
other hand, the strain strongly stimulated accumulation of
hybroxybenzoic acids being precursors of SA (Nawrocka
et al. 2017). In the present study, we assessed involvement
of different forms of PC including SA as well as VOC newly
identified in Tric hoderma pretreated plants in resistance in-
duction against R. solani. Among SA derivatives, TRS25 sig-
nificantly enhanced accumulation of MeSA, which is one of
the molecules involved in resistance signaling characteristic to
SAR (Park et al. 2007; Vogt 2010; Gao et al. 2014). Moreover,
the excess of SA seemed to be accumulated as SAGC, which
is considered as a storage form of this compound releasing SA
when necessary (Takatsuji et al. 2014) or volatile EHS, one of
natural pesticides rarely detected in plants (Yedida et al. 2003;
Vogt 2010 Singh et al. 2011; Oliveira et al. 2016). The in-
crease in the concentration of different SA derivatives in
TRS25 plants was accompanied by significant accumulation
of another VOC, β-cyclocitral, which recently was found to
up-regulate SA signaling (Lv et al. 2015). The accumulation
of all SA derivatives and VOC, mentioned above, was also
Fig. 3 Effect of TRS25 on relative expression of defense genes: aPR1,b
PR5,cPR4,anddPR12 in cucumber plants. Values represent the
means + SE from three independent experiments with six replicates
each. Statistical analysis of variance (ANOVA, P< 0.05) for each
parameter was followed by the Duncan multiple range post hoc test.
Respective significant differences were marked using letters a, b, c, and
d. Control plants (TRS25 nontreated, uninoculated with R. solani), Rs
plants (TRS25 nontreated, inoculated with R. solani), TRS25 plants
(TRS25 pretreated, uninoculated with R. solani), and TRS25 + Rs
plants (TRS25 pretreated, inoculated with R. solani)wereusedinthe
study
Involvement of metabolic components, volatile compounds, PR proteins, and mechanical strengthening in... 367
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
observed in TRS25 + Rs plants with significantly reduced
R. solani-induced disease symptoms. Moreover, in these
plants, simultaneous generation of unsaturated fatty acid de-
rivatives including Z-3-hexanal, Z-3-hexenol, and E-2-
hexenal was detected. In correlation with other VOC includ-
ing SA derivatives, these compounds might be among the
fastest signaling molecules able to directly elicit or prime plant
systemic defense response and resistance. According to Scala
et al. (2013), VOC signals seem to be faster than vascular
signal molecules and can also reach organs not directly con-
nected through vascular system. Thus, in this model, vascular
and airborne signals may act synergistically to ensure optimal
resistance in distal plant parts. Moreover, the present VOC,
especially E-2-hexenal and Z-3-hexenol with antimicrobial
activity, might prevent microbes from invading plants as well
as participate in stronger lignification of plant tissues (Scala
et al. 2013). All the mentioned VOC together with H
2
O
2
may
be involved in resistance signaling resulting in enhanced ex-
pression of PR proteins (Mathys et al. 2012; Brotman et al.
2013; Scala et al. 2013; Gao et al. 2014;Lvetal.2015;De
Palma et al. 2016;Adrianetal.2017). Since the genes
encoding PR proteins may be up-regulated by different sig-
naling molecules, their expression is considered as a marker of
the type of induced resistance (Yedidia et al. 2000;Shoresh
et al. 2005; Vinale et al. 2008). In the present study, the pro-
tection of cucumber against R. solani was effective in the
plants in which up-regulation of SAR-related PR1 and PR5
genes was accompanied by increase in the expression of ISR
gene PR4 in TRS25 + Rs plants, while this gene expression
did not increase in TRS25 plants. On the other hand, up-
regulation of PR4, unaccompanied by over-expression of
other tested genes in Rs plants neither decreased plant suscep-
tibility to R. solani attack nor induced even partial plant resis-
tance. The complex network of resistance inducers including
SA derivatives and unsaturated fatty acid derivatives present
in TRS25 + Rs plants may explain the enhanced expression of
genes characteristic of two kinds of systemic resistance, i.e.,
ISR/SAR. It is suggested that some of the genes induced by
unsaturated fatty acid derivatives including E-2-hexenal and
Z-3-hexenal are also induced by JA in Arabidopsis (Scala
et al. 2013). Thus, one could suggest that MeSA, EHS, and
β-cyclocitral participated in the up-regulation of PR1 and PR5
genes and SA-dependent response while Z-3-hexanal, Z-3-
hexenol, and E-2-hexenal promoted the up-regulation of
PR4 and JA-dependent responses that help cucumber plants
to counteract R. solani. Such stimulation was reported for Z-3-
hexenal and Z-3-hexenol which induced expression of several
defense JA-related genes in Arabidopsis such as defensin 1.2
(PDF1.2) and PR-3 (chitinase B) protecting the plant against
B. cinerea (Kishimoto et al. 2006). Moreover, it is possible
that the mentioned compounds may be accompanied by other
signaling VOC from the groups of unsaturated fatty acid de-
rivatives and PC; however, additional studies are necessary to
determine them. At the moment, the observed combined type
of gene up-regulation might be similar to the cross-TISR char-
acterized by Perazzolli et al. (2012) and Levy et al. (2015)in
Trichoderma harzianum-related protection of plants against
Plasmopara viticola,B. cinerea, and Podosphaera xanthii
which included SAR- and ISR-related gene expression includ-
ing PR1,PR2,andPR4 up-regulated in the presence of path-
ogens. The studied genes encode proteins, which may partic-
ipate in plant defense response, cell wall reinforcement, and
Fig. 4 The cross section of the
cucumber shoot (a) and root (b)
tissues. Fluorescent confocal
microscopy was used to detect
callose deposition in the control
and TRS25 plants. Abbreviations:
iph internal phloem, eph external
phloem, x xylem. The white
arrows indicate light green
fluorescence of callose in the cells
separating internal phloem from
the shoot pith in the cucumber
shoot vascular bundles (a)andin
tissue between xylem vessels of
adjacent vascular bundles in roots
(b)
368 J. Nawrocka et al.
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Fig. 5 The cross section of the
cucumber shoot (a,b,c), root (d),
and leaf (e) tissues. Fluorescent
confocal microscopy was used to
detect lignin deposition in the
control and TRS25 plants.
Abbreviations: iph internal
phloem, eph external phloem, x
xylem, nd nondetected, ep
epidermis, co collenchyma. The
white arrows indicate green
fluorescence of lignin detected in
cells surrounding of external
phloem in vascular bundles in the
cucumber shoot (a,b), in the
walls of epidermis and
collenchyma cells in the
cucumber shoot (c), and in
epidermis of leaves (e)
Involvement of metabolic components, volatile compounds, PR proteins, and mechanical strengthening in... 369
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direct protection against R. solani as it was presented previ-
ously for plants interacting with Trichoderma hamatum and
T. harzianum (Alfano et al. 2007; Tucci et al. 2011;Mathys
et al. 2012).
When plants are exposed to pathogens, simultaneously
to biochemical and molecular response, the mechanical
strengthening of plant tissues may contribute to pathogen
spread restriction (Chowdhury et al. 2014; Rao et al. 2015).
Plant defense response and resistance against fungal path-
ogens including R. solani was found to partly result from
stimulation of cell wall fortification by formation of lignin
and callose in plants (Yedidia et al. 2000; Solanki et al.
2011; Martínez-Cortés et al. 2012; Nikraftar et al. 2013).
Simultaneous involvement of peroxidase, chitinase, and
lignin formation in protection of tomato and rice against
R. solani was described by Taheri and Tarighi (2012).
Moreover, the accumulation of newly formed callose be-
yond the sites of fungal penetration in the epidermal and
cortical cell walls in the roots and leaves of cucumber plants
treated with T. harzianum was found previously by Yedidia
et al. (2000). In the present study, the contents of lignin and
callose were strongly enhanced in the plants pretreated with
TRS25 both in the absence and presence of R. solani.These
components were detected in the cucumber tissues, which
had no direct contact with TRS25 and R. solani. Since in the
previous studies enhancement of structural barriers was
mainly analyzed and described in the area of fungal influ-
ence (Chowdhury et al. 2014; Rao et al. 2015; Schneider
et al. 2016), we focused on systemic location of callose and
lignin in the whole plant. The microscopic analyses showed
that main localizations of these compounds were observed
in the area of vascular bundles and dermal tissues in cucum-
ber shoot, root, and leaves where they were localized in
different cells. In the cucumber shoot, callose deposited as
punctate distribution patterns seemed to be integral to spe-
cialized, separated cells which protected vessels at the side
of internal phloem. Lignin, which was most likely produced
by SPX from accumulated PP units, was distributed regu-
larly in the line of cell walls of an outer layer of external
phloem in the vascular bundles. The lignin layer was not
detectable in the control plants. The two compounds
seemed to act together also in the vascular bundles of roots
where lignin formed the xylem vessels, and callose was
localized in separate cells between adjacent vascular bun-
dles. Together, the compounds formed mechanical layer
protecting phloem and xylem from both sides not
restricting growth of other cells. They also protected the
walls of epidermis and collenchyma cells in shoots and
leaves from the outside environment and they made organs
more flexible and resilient, helped them maintain the cor-
rect position, and protected deeper layers of assimilation
cells. The obtained results suggest that callose and lignin
depositions might not only be involved in protection of
cucumber plants against spread of R. solani-induced dis-
ease and against plant cell degradation by enzymes and
toxins released by the pathogen as suggested previously
(Djonovićet al. 2007; Huang et al. 2010; Spoel and Dong
2012; Doorn and Ketsa 2014), but simultaneously, they
might play important role in plant growth promotion by
reinforcement of vascular system making water and nutri-
ent transport more effective.
Conclusions
Taken together, the study showed that T. atro vi ri de TRS25
strain was effective in growth promotion and in reduction
of R. solani-induced disease in cucumber plants. The sys-
temic disease suppression was not related to direct inhibi-
tion of R. solani growth and proliferation by TRS25 but
to stimulation of multilayer TISR. Treatment of the cu-
cumber roots with TRS25 induced biochemical, molecular,
and structural changes, which are important in plant de-
fense responses and resistance. In TRS25-induced TISR,
systemic enhancement of defense enzymes including
GPX, SPX, PAL, and PPO accompanied by accumulation
of H
2
O
2
and PC as well as decrease in lipid peroxidation
was detected. To the best of our knowledge, the important
role of accumulation of VOCs including EHS and β-
cyclocitral and of unsaturated fatty acid derivatives, that
is, Z-3-hexanal, Z-3-hexenol, and E-2-hexenal as well as
SA storage as SAGC was observed for the first time in
plants protected by Tr ichoderma against R. solani.Weput
forward a hypothesis that together with MeSA, all these
compounds may enhance expression of SAR- and ISR-
related genes leading to induction of mixed type of
TISR. Moreover, TRS25 inducing system of callose and
lignin deposition promoted mechanical plant strengthening.
The in-depth microscopic analysis revealed that the detect-
ed compounds protected vascular system both at internal
and external phloem sides in cucumber shoots. Lignin
formed the wall thickening xylem vessels, and callose
was localized in separate cells between adjacent vascular
bundles in roots. These compounds protected cells and
dermal tissues in cucumber roots, shoots, and leaves
against R. solani and made them more flexible and resil-
ient, which helped to maintain turgor and contributed to
better nutrition and hydration of plants. The growth pro-
motion coupled with systemic mobilization of biochemical,
molecular, and structural response might be positively in-
volved in multilayer protection of cucumber plants against
R. solani activated by TRS25. Based on the results,
TRS25 strain has potential to be an effective biocontrol
agent for R. solani-induced disease management in
cucumber plants.
370 J. Nawrocka et al.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Acknowledgements The authors are grateful to Ms. M. Fronczak for
her linguistic correction of themanuscript, to Ms. M. Wielanek for help in
HPLC analyses, and to Mr. Andrzej Kaźmierczak for substantive support
in microscopic techniques. This study was conducted as part of the pro-
ject BPolish Trichoderma strains in plant protection and organic waste
management^under Priority 1.3.1, co-financed by the European Union
through the European Regional Development Found within the
Innovative Operational Program, 2007–2013, Project No. UDA-
POIG.01.03.01-00-129/09-00.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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